Polyhydroxyalkanoate production method

ABSTRACT

Provided are processes for the production and high efficiency processing of polyhydroxyalkanoates (PHA) from carbon sources comprising carbon-containing gases or materials.

RELATED APPLICATIONS

This application is the U.S. National Phase application under 35 U.S.C.§371 of International Application No. PCT/2012/028210, filed on Mar. 8,2012, which claims the benefit of U.S. Provisional Application No.61/450,512, filed Mar. 8, 2011, the disclosure of each of which isincorporated by reference herein.

BACKGROUND

1. Field of the Invention

Embodiments of the invention relate to an improved process for theproduction and processing of polyhydroxyalkanoates, and specifically toa process for the production of polyhydroxyalkanoates fromcarbon-containing gases and materials.

2. Description of the Related Art

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters that serve ascarbon and energy storage vehicles in microorganisms. PHAs arebiodegradable in both aerobic and anaerobic conditions, arebiocompatible with mammalian tissues, and, as thermoplastics, can beused as alternatives to fossil fuel-based plastics such aspolypropylene, polyethylene, and polystyrene. In comparison topetrochemical-based plastics, which are neither biodegradable nor madefrom sustainable sources of carbon, PHA plastics afford significantenvironmental benefits.

The utilization of food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems is often regarded asthe most efficient and economical platform for PHA production.Specifically, sugar-based PHA production processes are capable ofgenerating high density fermentation cultures and high PHA inclusionconcentrations, and, by maximizing the cell culture density and PHAinclusion concentration therein, it is believed that carbon, chemical,and energy efficiencies are also maximized. For example, comparing a lowcell and PHA concentration process to a high cell and PHA concentrationprocess, a low concentration process requires significantly more, pergiven unit of PHA-containing biomass, i) energy for dewatering cellsprior to PHA extraction treatment, ii) liquid culture volume, andassociated chemicals, mixing energy, and heat removal energy, and iii)both energy and chemicals for separating PHA from biomass. Accordingly,whereas the sugar-based genetically-engineered microorganism PHA processyields maximized cell densities and PHA concentrations relative to lowconcentration processes, it is also regarded as the most carbon,chemical, energy, and, thus, cost efficient PHA production method.

Unfortunately, despite these maximized efficiency advantages,sugar-based PHA production remains more expensive than fossil fuel-basedplastics production. Thus, given the apparent efficiency maximization ofthe high density sugar-derived PHA production process, PHAs aregenerally considered to be unable to compete with fossil fuel-basedplastics on energy, chemical, and cost efficiency.

SUMMARY

Despite the environmental advantages of PHAs, the high cost of PHAproduction relative to the low cost of fossil fuel-based plasticsproduction has significantly limited the industrial production andcommercial adoption of PHAs.

To reduce the carbon input cost of the PHA production process,carbon-containing industrial off-gases, such as carbon dioxide, methane,and volatile organic compounds, have been proposed as an alternative tofood crop-based and plant-based sources of carbon. In addition to thewide availability and low cost of carbon-containing gases,carbon-containing gases also do not present the environmental challengesassociated with food crop and plant-derived sources of carbon.Specifically, whereas food crop- and plant-based carbon substratesrequire land, fertilizers, pesticides, and fossil fuels to produce, andalso generate greenhouse gas emissions during the course of production,carbon-containing off-gases do not require new inputs of land,fertilizers, or pesticides to generate. Thus, on both an economic andenvironmental basis, the utilization of carbon emissions for theproduction of PHA would appear to offer significant advantages oversugar-based PHA production processes.

Unfortunately, the fermentation of carbon-containing gases presentstechnical challenges and stoichiometric limitations that have, in thepast, rendered the gas-to-PHA production process significantly moreenergy and chemical intensive, and thus more costly, than the foodcrop-based PHA production process. Specifically, these technicalchallenges and stoichiometric limitations include: low mass transferrates, low microorganism growth rates, extended polymerization times,low cell densities, high oxygen demand, and low PHA cellular inclusionconcentrations. Whereas sugar-based fermentation systems have theability to generate high cellular densities and PHA inclusionconcentrations, based on cell morphology and mass transfer constraints,carbon-containing gas-based fermentation processes typically generate10-50% of the biomass and intracellular PHA inclusion concentrationsachieved in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon injection, optionaloxygen injection, and culture mixing, as well as downstream PHApurification, significantly exceeds the energy-to-PHA ratio required forsugar-based PHA production methods, thereby rendering theemissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based PHAs.

In light of the potential environmental advantages and carbon costefficiencies of utilizing carbon-containing gases as a source of carbonfor PHA production, there exists a significant need to reduce theenergy, chemical, and carbon input-to-PHA output ratio in a carbonemissions-based PHA production system, and thereby render carbongas-derived PHA economically competitive with petrochemical-basedplastics.

Thus, in several embodiments, the present invention relates to a novelprocess for the conversion of carbon-containing gases into PHAs atpreviously unattainable energy and carbon PHA conversion ratios.

In some embodiments, the invention also relates to a process thatgenerates a carbon emissions-based PHA material that is cost-competitivewith both food crop-based PHAs and fossil fuel-based thermoplastics.

While PHAs are widely considered to be noncompetitive with fossilfuel-based plastics on energy, chemical, and cost efficiency, severalembodiments of the invention relate to a process for producing PHAs fromcarbon-containing gases that yields unexpectedly improved energy,carbon, chemical, and cost efficiencies over sugar-based PHA productionmethodologies.

There is provided, in several embodiments, a method for enhancingpolyhydroxyalkanoate (PHA) generation in a methanotrophic culture byreducing copper concentrations to effect particulate methanemonooxygenase (pMMO) production comprising

contacting a culture of methanotrophic microorganisms with a mediumcomprising copper, one or more additional nutrients, and acarbon-containing gas that can be metabolized by the culture, incubatingthe culture in the medium to cause growth of the culture, inducing aselection pressure in the culture to transform the culture into aculture that generates PHA preferentially through pMMO by: (i) reducingthe concentration of copper in the medium to cause production of solublemethane monooxygenase (sMMO) and/or particulate methane monooxygenase bythe culture, wherein the concentration of copper causes the productionof sMMO in some methanotrophic microorganisms; (ii) reducing theconcentration of one or more the nutrient in the medium to cause theculture to generate PHA from the carbon-containing gas using the sMMO orthe pMMO, wherein PHA is generated by pMMO at a greater rate as comparedto sMMO, (iii) returning the culture to the growth conditions, whereinmicroorganisms having higher intracellular concentrations of pMMO andPHA grow at a greater rate as compared to those with lower intracellularpMMO and PHA concentrations; and (iv) repeating steps (ii) and (iii),wherein the repetitions selectively favor growth of microorganisms thatproduce PHA via pMMO, thereby facilitating the pMMO-mediated productionof PHA at reduced copper concentrations, and resulting in a culturecomprising essentially only microorganisms that use pMMO to produce PHA.

There is also provided a method for enhancing particulate methanemonooxygenase (pMMO) mediated polyhydroxyalkanoate (PHA) generationcomprising contacting a culture of methanotrophic microorganisms with amedium comprising copper, nitrogen, one or more additional nutrients,and a carbon-containing gas that can be metabolized by the culture,incubating the culture in the medium to cause growth of the culture; andtransforming the culture into a culture that generates PHApreferentially through pMMO by: (i) reducing the concentration of copperin the medium to cause production of soluble methane monooxygenase(sMMO) and particulate methane monooxygenase by the culture, wherein theconcentration of copper favors the production of sMMO; (ii) reducing theconcentration of nitrogen to cause the culture to generate PHA from thecarbon-containing gas using the sMMO or the pMMO, wherein PHA isgenerated by pMMO at a more rapid rate as compared to sMMO, resulting ina portion of the microorganisms having higher intracellularconcentrations of PHA as compared to those using sMMO; and (iii)returning the culture to the growth conditions, wherein themicroorganisms having higher intracellular concentrations of PHA grow ata greater rate as compared to those with lower intracellular PHAconcentrations; and (iv) repeating steps (ii) and (iii), wherein therepetitions selectively favor growth of microorganisms that produce PHAvia pMMO, thereby facilitating the pMMO-mediated production of PHA.

There is also provided, in several embodiments, a method for convertinga carbon-containing material into a polyhydroxyalkanoate (PHA), themethod comprising (a) contacting a culture of methanotrophicmicroorganisms with a medium comprising copper, one or more additionalnutrients, and a carbon-containing material that can be metabolized bythe culture, thereby inducing growth of the culture (b) reducing orcontrolling the concentration of copper in the medium to cause at leasta portion of the culture to produce soluble methane monooxygenase (sMMO)and/or particulate methane monooxygenase (pMMO), wherein the copperconcentration can cause one or more methanotrophic microorganism toproduce sMMO, (c) reducing the concentration of at least one of thenutrients in the medium to cause at least a portion of the culture toutilize the produced sMMO or the produced pMMO to convert the carbonfrom the carbon-containing material into PHA, (d) repeating steps (a)through (c) a plurality of times, wherein the portion of the cultureutilizing the pMMO to generate PHA grows and/or generates PHA at agreater rate than the portion of the culture utilizing the sMMO togenerate PHA, thereby inducing a selective pressure in the cultureresulting in a culture comprising essentially only microorganisms thatuse pMMO to produce PHA.

In several embodiments, the induced selection pressure selects formicroorganisms in which the presence and/or expression of the geneencoding the sMMO is reduced. This is unexpected, in severalembodiments, as the copper concentrations are such that sMMO productionwould typically be favored. When employed in combination with periods ofgrowth, PHA production, and growth, the selection pressure unexpectedlyand advantageously allows those microorganisms operating through thepMMO pathway to become grow more quickly, thereby outcompeting many, ifnot all, organisms using an alternate MMO pathway. In severalembodiments, as the growth-polymerization-growth cycles are repeated inconjunction with the existence of conditions favoring pMMO metabolism,the expression of the gene encoding the soluble methane monooxygenaseenzyme is reduced. Thus, the growth advantage of those microorganismsutilizing P MMO provides, in each cycle, at least a marginal gain withrespect to the pMMO-utilizing microorganisms increasing their percentagecontribution to the overall demographics of the culture. With eachsuccessive round, pMMO-utilizing microorganisms outcompete thesMMO-utilizing microorganisms, thereby eventually being the primary, ifnot only, microorganism in the culture. As a result this process, the“weaker” sMMO-utilizing microorganisms will be reduced in number, and insome cases may undergo an evolutionary-pressure induced change toreduce/eliminate sMMO expression in favor of pMMO.

In additional embodiments, alternative methods may be used to achieve areduction in the presence and/or expression of the sMMO gene and/orprotein. For example, as disclosed herein genetically modifiedmicroorganisms may be used in certain embodiments. Thus, prior to aculturing process in which PHA would be produced, in some embodiments,microbiological techniques are used to excise the genetic materialencoding sMMO from the genome of the microorganism. Similarly,microorganisms having the genetic material for sMMO could be culturedwith (e.g., bred with) an alternative strain of microorganisms havinggenetic material for a metabolically favorable (e.g., more active) pMMO.After successive rounds of crossbreeding, microorganisms are selectedbased on a combination of limited sMMO expression and robust highactivity pMMO expression. In additional embodiments, methods can be usedto suppress one or more of the expression where the activity of the sMMOenzyme, rather than manipulating the microorganism on genomic level. Forexample, RNA interference or antisense RNA could be used to suppressexpression and/or function of sMMO. Similarly site directed mutagenesiscould be used such that a microorganism would produce a mutant (e.g.,nonfunctional or minimally functional) sMMO. Site directed mutagenesiscan also be employed to introduce a specific mutation in the sMMO genesuch that a truncated and/or nonfunctional (or reduced function) S MMOis produced. For example, the introduction of mutation that results inearly placement of a stop codon in the sMMO gene would result in atruncated, and likely nonfunctional enzyme (if any enzyme and all werein fact be produced). In addition to, or in place of such approaches,inducible or repressible promoters could be employed such that sMMOwould only be expressed under certain conditions (e.g., the addition ofan inducing agent to the culture media) or alternatively would normallybe repressed absent the removal of repressing agent from the media. Akinto gene therapy approaches, microorganisms could be modified to carryexogenous DNA that produces a repressor of sMMO function (oralternatively, a promoter of pMMO function).

In some embodiments, the actual and/or bioavailable concentration of thecopper in the medium is reduced to a concentration less than about 0.001mg/L, less than about 0.5 micromolar, less than about 0.1 mg per dryweight gram of the microorganisms, less than about 100 mg/L, or lessthan about 1 mg/L. In some embodiments, the concentration of copper ismaintained at the reduced level. In some embodiments, the copperconcentration is returned to initial concentrations. In severalembodiments, reduction of one or more additional nutrient comprises asubstantial depletion of nitrogen from the media. Substantial depletioncomprises, in some embodiments, depletion of nitrogen concentrations byabout 50%, about 60%, about 70%, about 80%, about 90%, about 95%, ormore. In other embodiments, depending on the metabolic status of theculture, smaller reductions in nitrogen, another nutrient in the media,or combinations thereof may be used to effect PHA polymerization.

In several embodiments, the carbon-containing gas that the culture usesas a source of carbon to generate PHA comprises methane. In someembodiments, the carbon-containing gas comprises carbon dioxide. In someembodiments, the carbon-containing gas comprises one or more volatileorganic compounds. In some embodiments, one or more of methane, carbondioxide, volatile organic compounds, and other gasses or compounds maybe present in the carbon-containing gas.

In some embodiments, the carbon source need not be a gas. Acarbon-containing material comprising a non-gaseous material (eitheralone or in combination with a gas) may also be used in certainembodiments. For example, in some embodiments, the non-gaseous materialcomprises one or more volatile fatty acids, methanol, acetate, acetoneor acetic acid, formate, formaldehyde or formic acid, propane, ethane,or combinations thereof (or in combination with other metabolizablecarbonaceous materials). In addition, in some embodiments, thenon-gaseous material comprises microorganism biomass removed from onepolymerization stage and added to the medium in a subsequent growthstage.

Advantageously, the methods disclosed herein allow production PHA athigh intracellular concentrations. The concentrations, in someembodiments, are at least 70% of the dry biomass weight of themicroorganisms. In some embodiments, the concentrations are particularlyadvantageous because the high intracellular PHA concentration enablesmore rapid growth of those microorganisms. Thus, a feed-forward cycleexists, in some embodiments, in that a microorganism that is selectedfor by the induced pressures produces PHA more rapidly through the pMMOpathway, thereby having a greater concentration of PHA, which furtherimparts the ability to grow more rapidly (vis-à-vis those microorganismswith lower PHA concentrations, such as those utilizing the sMMOpathway). As the process is repeated, the pMMO-utilizing microorganismspossess a growth advantage at (at least) two stages in the process,thereby allowing their dominance in the culture. As such, in severalembodiments, the culture comprising essentially only microorganisms thatuse pMMO to produce PHA comprises a culture wherein over 50% of theculture uses pMMO to produce PHA. Additionally, in several embodiments,the culture comprising essentially only microorganisms that use pMMO toproduce PHA comprises over 80% of the culture. In several embodiments,the microorganisms in the culture are from the genus methylocystis.

In addition, several embodiments, the repeated cycling between growth,copper reduction to favor pMMO production, PHA production and back togrowth also favors the production of microorganisms that possess thegenetic material encoding the ethylmalonyl-CoA pathway. Advantageously,this pathway allows, in some embodiments, additional carbon sources tobe used by the culture, thereby broadening the range of substrates thatcan eventually be used as substrates for the generation of PHA.

A method for converting a carbon-containing material into apolyhydroxyalkanoate is also provided in several embodiments, the methodcomprising: (a) contacting a methanotrophic culture with a mediumcomprising one or more nutrients and a carbon-containing material thatcan be metabolized by the culture, (b) controlling the concentration ofthe one or more nutrients in the medium to cause at least a portion ofthe culture to produce sMMO and/or pMMO, (c) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA; (d) repeating steps (a) through (c) a pluralityof times.

In several embodiments, the nutrient of step (b) and the nutrient ofstep (c) are the same nutrient, while in other embodiments, the nutrientof step (b) and the nutrient of step (c) are different nutrients. In oneembodiment, the nutrient is selected from the group consisting ofcopper, methane, oxygen, phosphorus, potassium, magnesium, boron,sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof.

Also provided for in several embodiments herein is a method forconverting a carbon-containing material into a polyhydroxyalkanoate(PHA), the method comprising: (a) providing a microorganism culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture, wherein the genetic material encoding the ethylmalonyl-CoApathway is present in the one or more microorganisms, (d) controllingthe concentration of the one or more nutrient in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d).

In several embodiments, the nutrient is copper, and step (c) comprisesperiodically or permanently reducing the concentration of the copper inthe medium. In several embodiments, the actual and/or bioavailableconcentration of the copper in the medium to be less than about 0.001mg/L, less than about 0.5 micromolar, less than about 0.1 mg per dryweight gram of the microorganisms, less than about 100 mg/L, or lessthan about 1 mg/L. In several embodiments, controlling the actual and/orbioavailable concentration of the copper in the medium effects theproduction of microorganisms that do not possess the gene encodingsoluble methane monooxygenase or express sMMO at reduced amounts. Inother embodiments, the organisms, have the gene, but the enzyme is notexpressed.

In some embodiments, the nutrient is selected from the group consistingof copper, methane, oxygen, phosphorus, potassium, magnesium, boron,sodium, calcium, nitrogen, iron, carbon dioxide, and combinationsthereof, and step (c) comprises periodically or permanently increasing,reducing, or maintaining the concentration of the nutrient in the mediumto effect the production of microorganisms that do not possess the geneencoding soluble methane monooxygenase. In several embodiments, thenutrient of step (c) and the nutrient of step (d) are the same nutrient,while in other embodiments, the of nutrient of step (c) and the nutrientof step (d) are different nutrients.

In several embodiments, steps (a) through (d) are repeated a pluralityof times and the concentration of the one or more microorganisms in theculture increases as the steps (a) through (d) are repeated.

There is also provided, in several embodiments, a method for convertinga carbon-containing material into a polyhydroxyalkanoate (PHA), themethod comprising: (a) providing a microorganism culture, (b) providinga medium comprising one or more nutrient comprising a carbon-containingmaterial that can be metabolized by the culture, (c) controlling theconcentration of the one or more nutrient in the medium to cause thecellular replication of one or more microorganisms in the culture,wherein the genetic material encoding the ethylmalonyl-CoA pathway ispresent in the one or more microorganisms, and wherein the expression ofsoluble methane monooxygenase is reduced in the one or moremicroorganisms as compared to a first instance of step (a), (d)controlling the concentration of the one or more nutrient in the mediumto cause the culture to produce PHA, and (e) repeating steps (a) through(d).

Copper concentrations can be reduced, in several embodiments, asdiscussed herein, e.g., to concentrations less than about 0.001 mg/L,less than about 0.5 micromolar, less than about 0.1 mg per dry weightgram of the microorganisms, less than about 100 mg/L, or less than about1 mg/L. Also as discussed above, the nutrients can be the same in steps(b) and (c), or alternatively they may be different. In one embodiment,the nutrient of step (c) is selected from the group consisting ofmethane, oxygen, phosphorus, potassium, magnesium, boron, sodium,calcium, nitrogen, iron, carbon dioxide, and combinations thereof andthe nutrient of step (d) is selected from the group consisting ofmethane, oxygen, phosphorus, potassium, magnesium, boron, sodium,calcium, nitrogen, iron, carbon dioxide, and combinations thereof, butis not the same as the nutrient of step (c).

There is also provided, method for controlling the functionalcharacteristics of polyhydroxyalkanoate (PHA) produced by amethanotrophic culture exposed to methane and one or more non-methanematerials that influence the metabolism of the culture, the methodcomprising: (a) providing a methanotrophic culture in a mediumcomprising one or more nutrients, (b) controlling the concentration ofone or more of the nutrients in the medium to cause the culture toproduce soluble methane monooxygenase (sMMO) and/or particulate methanemonooxygenase (pMMO), (c) controlling the concentration of the one ormore nutrients in the medium to cause the culture to produce PHA, and(d) repeating steps (a) through (c), wherein the concentration of sMMOrelative to pMMO in step (c) is substantially the same in eachrepetition, wherein one or more functional characteristics of the PHAproduced by the culture are controlled in multiple PHA productionrepetitions, and wherein the culture selectively generatesmicroorganisms that synthesize PHA at high efficiency.

In several embodiments, the concentration of the sMMO relative to thepMMO in the culture is substantially the same in step (c) in at leasttwo consecutive repetitions. Advantageously, this consistency results ina more defined and predictable PHA. For example, in several embodiments,PHA produced by the culture exhibits substantially the same of one ormore of the following characteristics in one or more the repetitions:molecular weight, polydispersity, impact strength, elasticity,elongation, and/or modulus. As discussed above, a variety of carbonsources may be used. In some embodiments, non-methane sources are used,such as carbon dioxide, volatile organic compounds, volatile fattyacids, methanol, acetate, acetone, and acetic acid, formate,formaldehyde, and formic acid, propane, ethane, oxygen, acarbon-containing material that can be metabolized by the culture, orcombinations thereof.

In one embodiment, at least 60% of microorganisms in the culture produceonly the pMMO, while in one embodiment at least 60% of microorganisms inthe culture produce only the sMMO. In still additional embodiments, theculture comprises an equal concentration of the sMMO and the pMMO. Inone embodiment, the concentration of the sMMO is more than 2 timesgreater than the concentration of the pMMO in the culture. In oneembodiment, the concentration of the sMMO is more than 5 times greaterthan the concentration of the pMMO in the culture. In one embodiment,the concentration of the sMMO is more than 10 times greater than theconcentration of the pMMO in the culture. In other embodiments, theconcentration of the pMMO is more than 2 times greater than theconcentration of the sMMO in the culture. In one such embodiment, theconcentration of the pMMO is more than 5 times greater than theconcentration of the sMMO in the culture.

There is also provided a method for modifying the functionalcharacteristics of a polyhydroxyalkanoate (PHA) material, comprising thesteps of: (a) providing a PHA and a biomass, (b) subjecting the PHAand/or the biomass to a processing step in order to render the at leasta portion of the biomass miscible with the PHA, (c) combining the PHAand the biomass in a mixture to form a compound, (d) heating thecompound to between 50 degrees Celsius and 250 degrees Celsius, and (e)causing the biomass to effect a functional modification of the PHA,wherein the functional modification comprises plasticization,nucleation, compatibilization, melt flow modification, strengthening,and/or elasticizaton.

Advantageously, a variety of types of biomass may be used, for example,one or more of methanotrophic, autotrophic, and heterotrophic biomassare used in several embodiments. In one embodiment, the processingcomprises treating the PHA and/or the biomass with one or more of thetreatments selected from the group consisting of: heat, shear, pressure,solvent extraction, washing, filtration, centrifugation, sonication,enzymatic treatment, super critical material treatment, cellulardissolution, flocculation, acid and/or base treatment, drying, lysing,and chemical treatment.

The biomass may be present at varied concentrations, depending on theembodiment. For example in one embodiment, the biomass is present in thecompound at a concentration of more than 0.001%, while in otherembodiments, the biomass is present in the mixture at a concentration ofmore than 0.01%. In still other embodiments, depending on the make-up ofthe biomass and the characteristics of the PHA, greater or lesserconcentrations may also be used.

The functional characteristics of PHA may also be modified through themelting and cooling of the PHA polymer in the presence of adual-miscible biomass agent and a second polymer by a method provided inseveral embodiments herein, the method comprising, comprising the stepsof: (a) providing a first polymer, a biomass, and a second polymer,wherein the first polymer is a PHA, (b) subjecting the biomass to aprocessing step comprising heat, pressure, solvent washing, filtration,centrifugation, super critical solvent extraction, and/or shear, whereinthe processing step renders at least a portion of the biomass misciblewith the first polymer and the second polymer, (c) contacting the firstpolymer with the biomass and the second polymer to form a compound, (d)heating the compound to between 50 degrees Celsius and 250 degreesCelsius, thereby causing the biomass to effect a functional modificationof the first polymer, the second polymer, and the combination of thefirst polymer and the second polymer, wherein the functionalmodification comprises plasticization, nucleation, compatibilization,melt flow modification, strengthening, and/or elasticization.

The biomass may be present at varied concentrations, depending on theembodiment. For example in one embodiment, the biomass is present in thecompound at a concentration of more than 0.001%, while in otherembodiments, the biomass is present in the mixture at a concentration ofmore than 0.01%. In still other embodiments, depending on the make-up ofthe biomass and the characteristics of the PHA, greater or lesserconcentrations may also be used. Advantageously, a variety of types ofbiomass may be used, for example, one or more of methanotrophic,autotrophic, and heterotrophic biomass are used in several embodiments.

Also provided for herein is a method for the synthesis of apolyhydroxyalkanote (PHA) in a biomass material, comprising the stepsof: (a) providing a medium comprising a biomass capable of metabolizinga source of carbon, and (b) increasing the concentration of one or morenutrients in the medium to cause the biomass to synthesize PHA orincrease the synthesis rate of PHA relative to the synthesis rate ofnon-PHA material.

Advantageously, a variety of types of biomass may be used, for example,one or more of methanotrophic, autotrophic, and heterotrophic biomassare used in several embodiments.

In several embodiments, the step of increasing the concentration of anutrient in the medium causes a reduction in the rate of production ofnon-PHA biomass relative to the rate of production of the PHA in thebiomass. This relative increase in efficiency (e.g., the reduction ofmetabolic resources spent producing non-PHA biomass) advantageouslyincreases the PHA production capacity of the biomass material (e.g., byincreasing the overall output, increasing the per unit biomass output orconcentration).

In several embodiments, the nutrient is selected from the groupconsisting of: ethylenediaminetetraacetic acid (EDTA), citric acid,iron, copper, magnesium, manganese, zinc, chromium, nickel, boron,molybdenum, calcium, potassium, boron, methane, phosphorus, oxygen,nitrogen, carbon dioxide and combinations thereof.

There is also provided a method for the synthesis of apolyhydroxyalkanote (PHA) in a biomass material, comprising the stepsof: (a) providing a medium comprising a biomass and one or morenutrients, and (b) increasing the concentration of one or more of thenutrients in the medium to cause the biomass to metabolically synthesizePHA by using the biomass as a source of carbon for the production of thePHA. In several embodiments, the nutrient is selected from the groupconsisting of magnesium, iron, copper, zinc, nickel, chromium,phosphorus, oxygen, calcium, methane, carbon dioxide, hydroxyl ions,hydrogen ions, sulfate, nitrogen, and combinations thereof. Varioustypes of biomass may be used, for example, one or more ofmethanotrophic, autotrophic, methanogenic and heterotrophic biomass areused in several embodiments

Complementary to the methods, processes, and systems for PHA production,there is also provided herein a process for extracting apolyhydroxyalkanoate from a PHA-containing biomass slurry comprisingPHA, non-PHA biomass, and a liquid, comprising: a) controlling thepressure and temperature of the liquid to cause the non-PHA biomass tobecome soluble in the liquid, and b) reducing the amount of the liquidin the slurry.

Depending on the embodiment, the liquid in the slurry may be one or moreof methanol, water, carbon dioxide, or another a solvent, super criticalfluid, polymer, additive, plasticizer, and/or other fluid (e.g.,methylene chloride, acetone, methanol, sodium hypochlorite,hypochlorite, chloroform, or dichloroethane). Various pressures areused, depending on the embodiment, including pressure above atmosphericpressure and pressure below atmospheric pressure. In still additionalembodiments, phases or sequential changes between sub-atmospheric andsupra-atmospheric pressures are used. In conjunction with the pressure,temperatures may be varied, in some embodiments, ranging between 0 and250 degrees Celsius. In other embodiments, a broader range oftemperatures is used, for example −40 degrees Celsius to about 500degrees Celsius, including about −20, about −4, about 4, about 20, about37, about 100, about 150 about 200, about 300, or about 400 degreesCelsius. In one embodiment, the temperature is between 0 and 200 degreesCelsius and the pressure is between −30 mmHg and 30,000 psi. In severalembodiments, temperatures are maintained between 0 and 400 degreesCelsius, 20 and 80 degrees Celsius, 10 and 100 degrees Celsius, 5 and200 degrees Celsius, 100 and 200 degrees Celsius, or 0 and 300 degreesCelsius. It shall be appreciated that different combinations oftemperature and pressure may be used, depending on the makeup of theslurry, the concentration of PHA in the slurry and other factors. Alsodepending on the characteristics of the slurry and other factors, thestep of reducing the amount of the liquid in the slurry comprisesreducing the amount of the liquid in the biomass slurry before, during,and/or after the step (a).

Reduction in the amount of the liquid can be achieved in a variety ofways, used either alone or in combination (e.g., sequentially) such asfor example, centrifugation, filtration, distillation, spray drying,flash drying, lyophilization, air drying, and/or oven drying. Theseprocesses can reduce the amount of liquid in the slurry by about 0.1 toabout 100%. In several embodiments, the reduction is over 50%, over 90%,or over 95%. Which method is chosen depends, in some embodiments, on thestate of the slurry at that time. Some methods are more efficient atremoval of liquid that others, and as such, may be better suited toslurries with a higher liquid content.

In some, embodiments, it may be necessary to increase the amount ofliquid in the slurry (e.g., to effect a more efficient downstream step),which depending on the embodiment can be done before, during, and/orafter the step (a). Various amounts of liquid may be added, depending onthe embodiment, for example such that the solids concentration in theslurry of less than about 1 g/L, less than about 5 g/L, less than about100 g/L, less than about 250 g/L, less than about 500 g/L, less thanabout 750 g/L, less than about 1000 g/L. or about 1300 g/L.

There is also provided a method for improving the functionalcharacteristics of a polyhydroxyalkanoate (PHA) material, comprising thesteps of: (a) providing a PHA, a biomass, and a non-PHA polymer, (b)combining the PHA, the non-PHA polymer, and the biomass in a mixture toform a compound, (c) heating the compound to between 100 degrees Celsiusand 250 degrees Celsius. Depending on the embodiment, the biomass ispresent in the mixture at a concentration of between about 0.1 and about0.8%, between about 0.1 and about 20%, between about 0.1 and about 40%,between about 0.1 and about 60%, between about 0.1 and about 80%., andoverlapping ranges thereof.

In several embodiments the biomass is methanotrophic biomass, while inother embodiments, the biomass is autotrophic biomass, heterotrophicbiomass, in combination, alone or with methanotrophic and/ormethanogenic biomass.

In several embodiments, the non-PHA polymer is one or more of thefollowing: polypropylene, polyethylene, polystyrene, polycarbonate,Acrylonitrile butadiene styrene, Polyethylene terephthalate, Polyvinylchloride, Fluoropolymers, Liquid Crystal Polymers, Acrylic,Polyamide/imide, Polyarylate, Acetal, Polyetherimide, Polyetherketone,Nylon, Polyphenylene Sulfide, Polysulfone, Cellulosics, Polyester,Polyurethane, Polyphenylene oxide, Polyphenylene Ether, StyreneAcrylonitrile, Styrene Maleic Anhydride, Thermoplastic Elastomer, UltraHigh Molecular Weight Polyethylene, Epoxy, Melamine Molding Compound,Phenolic, Unsaturated Polyester, Polyurethane Isocyanates, Urea MoldingCompound, Vinyl Ester, Polyetheretherketone, Polyoxymethylene plastic,Polyphenylene sulfide, Polyetherketone, Polysulphone, Polybutyleneterephthalate, Polyacrylic acid, Cross-linked polyethylene, Polyimide,Ethylene vinyl acetate, Styrene maleic anhydride, Styrene-acrylonitrile,Poly(methyl methacrylate), Polytetrafluoroethylene, Polybutylene.Polylactic acid, Polyvinyl chloride, Polyvinyl acetate, Polyvinylacetate co-Polyvinylpyrrolidone, Polyvinylpyrrolidone, Polyvinylalcohol, Cellulose, Lignin, Cellulose Acetate Butryate, Polypropylene,Polypropylene Carbonate, Propylene Carbonate, Polyethylene, ethylalcohol, ethylene glycol, ethylene carbonate, glycerol, polyethyleneglycol, pentaerythritol, polyadipate, dioctyl adipate, triacetylglycerol, triacetyl glycerol-co-polyadipate, tributyrin, triacetin,chitosan, polyglycidyl methacrylate, polyglycidyl metahcrylate,oxypropylated glycerine, polyethylene oxide, lauric acid, trilaurin,citrate esters, triethyl citrate, tributyl citrate, acetyl tri-n-hexylcitrate, saccharin, boron nitride, thymine, melamine, ammonium chloride,talc, lanthanum oxide, terbium oxide, cyclodextrin, organophosphoruscompounds, sorbitol, sorbitol acetal, sodium benzoate, clay, calciumcarbonate, sodium chloride, titanium dioxide, metal phosphate, glycerolmonostearate, glycerol tristearate, 1,2-hydroxystearate, celluloseacetate propionate, polyepichlorohydrin, polyvinylphenol, polymethylmethacrylate, polyvinylidene fluoride, polymethyl acrylate,polyepichlorohydrin-co-ethylene oxide, polyvinyl idenechloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, and/or Polyvinylidene chloride.

There is also provided, in several embodiments, a method for thesynthesis of polyhydroxyalkanote (PHA) in a biomass material, comprisingproviding a medium comprising a biomass metabolizing a source of carbon,and increasing or maintaining above a minimum, the concentration of anelement in the medium to cause the biomass to synthesize PHA and/orincrease the synthesis rate of PHA relative to the synthesis rate ofnon-PHA material. In several embodiments the PHA is polyhydroxybutyrate(PHB), while in other embodiments other types of PHA are produced. Inseveral embodiments, the biomass is one or more microorganisms. In oneembodiment, the biomass comprises one or more recycled microorganisms(e.g., those that have already been processed through a PHA synthesisprocess). In one embodiment, the microorganisms have been processed toremove at least a portion of the PHA they produced in the priorsynthesis process.

In several embodiments, the increase in the concentration of an elementin the medium above a minimum concentration (or maintenance above thatminimum) causes a reduction in the concentration or production of sugar,lipids, nucleic acids, saccharides, polysaccharides, methanobactin,and/or pigments in the biomass relative to the concentration orproduction of PHA in the biomass. In several embodiments, the elementthat is increased or maintained is one or more of phosphorus, oxygen, ornitrogen. In additional embodiments, the element is one or more of EDTA,citric acid, iron, copper, magnesium, manganese, zinc, calcium,potassium, boron, methane, or carbon dioxide.

In still additional embodiments, the PHA synthesis rate is increasedrelative to the synthesis rate of PHA in the biomass in the absence ofthe increase or maintenance above a minimum the concentration of anelement in the medium.

There is also provided a method for the synthesis of polyhydroxyalkanote(PHA) in a biomass material, comprising the steps of: (a) providing amedium comprising a biomass and an element, and (b) maintaining above aminimum concentration or increasing the concentration of the element inthe medium to cause the biomass material to metabolically synthesize PHAat the expense of alternative biomass energy and/or carbon storagematerials.

In several embodiments, the element that is increased (or maintained) isone or more of phosphorus, oxygen, magnesium, calcium, copper, iron,methane, carbon dioxide, hydroxyl ions, hydrogen ions, or nitrogen. Insome embodiments, the amount of increase (or maintenance) that isrequired of one element is altered when more than one element ismanipulated. For example, if phosphorous is increased in combinationwith another element, the overall amount of phosphorous needed isreduced as compared to the addition of phosphorous alone (e.g., thecombination of elements potentiates the effect).

In several embodiments, the biomass comprises one or moremicroorganisms, such as for example, methanotrophic microorganisms,heterotrophic microorganisms, autotrophic microorganisms, methanogenicmicroorganisms, or combinations thereof.

More specifically, certain embodiments of the invention provide highefficiency, high density, high PHA concentration processes for theproduction of PHA from carbon-containing gases, comprising the steps of:(a) providing a microorganism culture comprising PHA-containing biomass,(b) removing a portion of the PHA-containing biomass from the culture,(c) extracting a portion of PHA from the removed culture to produceisolated PHA and PHA-reduced biomass, (d) purifying the isolated PHA,and (e) returning the PHA-reduced biomass to the culture to cause theculture to convert the carbon within the PHA-reduced biomass into PHA.In several embodiments, carbon output from the system is wholly orsubstantially only in the form of PHA.

In several embodiments, a system for using a microorganism culture toconvert a carbon-containing gas into PHA at high efficiencies isprovided. Microorganisms are cultured using a combination of one or morecarbon-containing gases and PHA-reduced biomass, or derivatives thereof,as sources of carbon to produce PHA-containing biomass. A portion of thePHA-containing biomass is then removed from the culture, and PHA isextracted from the removed PHA-containing biomass to createsubstantially PHA-reduced biomass and substantially isolated PHA.

Typically, PHA is present in the PHA-containing biomass of gas-utilizingmicroorganisms at concentrations in the range of about 5%-60%, andapproximately 40-95% of the PHA-containing biomass is discarded from thesystem following PHA extraction. In some cases, PHA is present ingas-utilization microorganisms in the range of about 1-90%, including atabout 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, or 90%, and approximately 10-99% of the PHA-containing biomassis discarded from the system following PHA extraction, including 99%,97%, 95%, 93%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%,20%, or 10% of the PHA-containing biomass. Rather than discarding theremaining, e.g., 40-95% of the PHA-reduced biomass, in one embodiment ofthe invention, the PHA-reduced biomass is returned back to themicroorganism culture to be regenerated as PHA by a microorganismculture capable of utilizing PHA-reduced biomass, or a derivativethereof, as a source of carbon for PHA production, thereby creating aregenerative closed-loop polymerization system. By using PHA-reducedbiomass as a source of carbon for PHA production in microorganismsgrowing as or in association with gas-utilizing microorganisms, PHA canbe produced from carbon-containing gases at surprisingly andunexpectedly improved carbon, energy, and chemical efficiencies, sincecarbon from carbon-containing gases that would otherwise be discarded isregenerated as PHA in a microorganism culture, and microorganisms thatproduce PHA from carbon-containing gases at low concentrations (e.g.,5-60% PHA by weight, or less than 70% PHA by weight) can, in someembodiments, be utilized to produce PHA at significantly increasedcarbon-to-PHA efficiencies. In some embodiments, the regeneration stepis repeated to form an essentially closed-loop system. Thus, in someembodiments, the carbon output from the system is at least 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% PHA In other words,at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95%, or 99% ofthe carbon entering the system is converted into PHA. In otherembodiments, 1-5%, 5-10%, 10-20%, 20-30%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% (and overlapping rangesthereof) of the carbon entering the system is converted to PHA. Byregenerating PHA-reduced biomass as PHA in a microorganism culture, thepercentage of carbon from a carbon containing gas that is converted toPHA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or greater than systems that do not employ theregenerative or closed-loop system disclosed herein. In someembodiments, the regeneration (e.g., return and/or recycling of thePHA-reduced biomass) step is repeated at least 2, 3, 4, 5, 6, 7, 8, 9,or 10 (or more) times. In some embodiments, the regeneration step isrepeated until at least 90% to 95% of the carbon input into the systemis converted into PHA. In some embodiments, the regeneration step isrepeated as many times as desired to reach a particular percentageconversion of carbon to PHA.

Several embodiments of the invention provide for the production of PHAfrom carbon-containing gases at previously unattainable energy, carbon,and chemical efficiencies by way of providing a microorganism culturecapable of metabolizing the carbon within both a carbon-containing gasand PHA-reduced biomass, manipulating the conditions of the culture tocause the culture to produce PHA, removing a portion of PHA-containingbiomass from the culture, extracting the PHA within the removedPHA-containing biomass to create substantially isolated PHA andsubstantially PHA-reduced biomass, returning the PHA-reduced biomass tothe culture and contacting the PHA-reduced biomass with the culture tocause the culture to metabolize the carbon within the PHA-reducedbiomass into PHA, and purifying the isolated PHA. Thus, an advantage ofseveral embodiments of the invention is the production of PHA fromcarbon-containing gases at significantly improved energy, carbon, andchemical efficiencies.

The process according to several embodiments disclosed herein yields arange of surprising benefits over current gas-based PHA productiontechnologies. To begin, whereas the cell density of gas-basedfermentation processes is traditionally limited by the mass transfer ordiffusion rates of one or more factors, such as light, oxygen, carbondioxide, methane, or volatile organic compounds, several embodimentsdisclosed herein enable the generation of cell densities thatsignificantly exceed cell densities attainable in other currentpractices (e.g., by more than 1%, 10%, 20%, 30%, 50%, 80%, 100% ormore), and thereby enables cost-efficient system mixing, aeration, heatcontrol, and dewatering. For example, current methane-based PHAproduction systems are known to be capable (based on cell morphology andmass transfer characteristics) of generating approximately 60 g/L ofbiomass with an overall PHA concentration of 55%, or 33 g/L PHA. Incontrast, in several embodiments of the invention, cell densities ofapproximately 135 g/L with an overall PHA concentration of 70%, or 94.5g/L PHA are generated in a methane-based PHA production system. In someembodiments, cell densities of approximately 10 g/L, 20 g/L, 30 g/L, 60g/L, 75 g/L, 100 g/L, 125 g/L, 135 g/L, 150 g/L or greater are achieved.In some embodiments, overall PHA concentration in such cultures rangesfrom approximately 1% to 20%, 20% to 30%, 30% to 55%, 55% to 60%, 65% to70%, 70% to 80%, 80% to 90%, or 95% or greater, (and overlapping rangesthereof) result. In several embodiments, such PHA concentration rangesrepresent significant, unexpected, and surprising improvements overtraditional processes, e.g., processes that are limited to low celldensities and/or PHA concentrations.

As a non-limiting example of the impact of this improvement on energyefficiency, the energy required, on an energy input-to-PHA output basis,to aerate, mix, and dewater a 135 g/L solution with a PHA concentrationof 70% by weight is 186% less than the energy required to aerate, mix,and dewater a 60 g/L microorganism solution comprising 40% PHA byweight. It shall be appreciated that variations in the energy efficiencygains based on the systems and processes disclosed herein may occur,depending on the culture conditions, the strain or organisms used, andthe concentration and/or purity of an initial gas stream or other carbonsource. In several embodiments, even modest increases in efficiency havesubstantial benefits. For example, the ability to efficiently use aninput gas having a low carbon concentration that would not otherwise beuseful in PHA production may prevent the release of such a gas into theenvironment and/or reaction of the gas with other atmospheric compounds,thereby reducing the adverse impact of the low carbon concentration gason the environment (e.g., destruction of ozone, greenhouse gas emission,pollution, etc.).

Additionally, whereas current gas-based PHA production systems producesignificant carbon losses as a result of the low PHA inclusionconcentrations of gas-utilizing microorganisms (e.g., a significantportion of carbon and energy input is lost as biomass), severalembodiments of the invention enable the generation of overall carboninput yield efficiencies approaching maximum substrate values; e.g.,100% carbon input-to-PHA yield, minus respiration and/or downstreamprocessing losses. In some embodiments, at least 5%, at least 10%, atleast 30%, at least 50%, at least 70%, or at least 90%, carboninput-to-PHA yield is achieved. It is one important advantage of severalembodiments of the invention that maximum carbon yield efficiencies areunexpectedly and surprisingly generated in a PHA production systememploying gas-utilizing microorganisms, and particularly, in someembodiments, in PHA production systems employing gas-utilizingmicroorganisms that produce low biomass and/or PHA inclusion densities.

In some embodiments, the microorganism culture is a mixed culture,comprising heterotrophic microorganisms, methanotrophic microorganisms,autotrophic microorganisms, bacteria, yeast, fungi, algae, orcombinations thereof. In other embodiments, the microorganism culturemay be one or more cultures (e.g., a plurality of cultures). In someembodiments, the cultures are grown in one or more bioreactors. In someembodiments, the bioreactors utilize one or more culture conditions,including both aerobic and anaerobic conditions. In some embodiments,the microorganism culture converts PHA-reduced biomass to methane in ananaerobic process and subsequently to PHA in an aerobic process, suchthat PHA-reduced biomass is first anaerobically metabolized to methaneand then used as methane to produce biomass and PHA in a methanotrophicculture.

In several embodiments, at least part of the microorganism culture is amixed culture capable of metabolizing carbon-containing gases, includingmethane, carbon dioxide, greenhouse gases, and/or various other volatileorganic compounds, into biomass and/or PHA. In some embodiments, themicroorganism culture comprises a two phase system of anaerobic andanaerobic/aerobic metabolism, whereby carbon-containing gas is producedin a first substantially anaerobic phase and subsequently converted intoPHA in a second phase, wherein the microorganism culture in the firstphase is substantially anaerobic and the culture in the second phase iseither anaerobic or aerobic, wherein the two phases may be operated inone single vessel or in multiple vessels.

In some embodiments, at least one or more of the microorganisms arecontacted with artificial and/or natural light during one or more stepsof the methods disclosed herein.

In some embodiments, at least one of more of the microorganisms iscontacted with dissolved oxygen during one or more steps of the methodsdisclosed herein.

In some embodiments, at least one of more of the microorganisms iscultured at atmospheric, sub-atmospheric, or above-atmosphericpressures.

In some embodiments, at least one of more of the microorganisms canutilize only a carbon-containing gas as a source of carbon.

In several embodiments, at least one or more of the microorganisms canutilize carbon derived from a PHA-reduced biomass as a source of carbon.In other embodiments, at least one or more of the microorganisms is aheterotrophic microorganism capable of converting PHA-reduced biomassinto, carbon dioxide, oxygen, biomass, and/or PHA.

In several embodiments, at least one or more of the microorganisms arecultured using carbon derived from both a carbon-containing gas and aPHA-reduced biomass.

In some embodiments, the microorganism culture is a pure culture. Insome embodiments, the cultures are maintained in semi-sterile or sterileconditions.

In some embodiments, the microorganism culture is a mixed, non-sterileculture, including a naturally equilibrating consortium ofmicroorganisms.

In several embodiments, the microorganism culture is at least partiallycomprised of genetically engineered microorganisms.

In some embodiments, the microorganism culture is a mixed culturecomprising a combination of naturally occurring and geneticallyengineered microorganisms.

In several embodiments, the PHA is removed from the microorganismculture by solvent extraction, including solvent extraction attemperatures ranging from 0° C. to 200° C. and at pressures ranging from−30 psi to 30,000 psi.

In several embodiments, the PHA is removed from the microorganismculture through the utilization of ketones, alcohols, and/or chlorinatedsolvents.

In several embodiments, the PHA is removed from the microorganismculture by hypochlorite digestion and/or chlorine-based solventextraction.

In several embodiments, the PHA is removed from the microorganismculture by supercritical carbon dioxide extraction.

In several embodiments, the PHA is removed from the microorganismculture by protonic non-PHA cell material dissolution.

In several embodiments, the PHA is partially removed from themicroorganism culture to create a PHA-rich phase and a PHA-poor phase.

In several embodiments, the PHA is removed from the microorganismculture to render the PHA substantially free of non-PHA material,including substantially 5%, 10%, 20%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80-90%, 90-99% or more pure PHA by weight.

In several embodiments, the PHA is removed from the microorganismculture by manipulating the pH of the microorganism culture.

In certain embodiments, at least one or more of the microorganisms arecontacted with methane, carbon dioxide, oxygen, and/or a combinationthereof.

In several embodiments, multiple culture vessels are employed, such thatmicroorganism growth, PHA synthesis, PHA-reduced biomass metabolism, andPHA removal are carried out in separate vessels.

In other embodiments, microorganism growth, PHA-reduced biomassmetabolism, and PHA synthesis occurs in a single vessel.

In still other embodiments, microorganism growth and PHA synthesis occurin a single vessel and PHA extraction is carried out in one or moreseparate vessels.

In several embodiments of the process as disclosed herein, PHA synthesisis regulated by manipulating, increasing, decreasing, maintaining belowa maximum threshold, or maintaining above a minimum threshold theconcentration of a material in the process, wherein the material is oneor more of oxygen, methane, carbon dioxide, nitrogen, phosphorus,copper, iron, manganese, carbon, magnesium, potassium, cobalt, aluminum,sulfate, chlorine, boron, citric acid, and EDTA.

In several embodiments, the microorganism culture comprises one or morestrains of microorganisms collectively capable of converting the carbonwithin a carbon-containing gas into cellular biomass and the carbon fromcellular biomass or methane into PHA.

In several embodiments, the microorganisms are subjected to filtration,centrifugation, settling, and/or density separation.

In several embodiments, the isolated PHA and/or the PHA-reduced biomassis subjected to filtration, centrifugation, settling, and/or densityseparation.

In some embodiments, the processes disclosed herein further comprisewashing the recovered PHA with water or other liquid-based agents orsolvents to purify the PHA.

In several embodiments, the process further comprises oxidizing therecovered PHA to purify the PHA.

In several embodiments, the process further comprises drying therecovered PHA to remove volatiles such as water and/or one or moresolvents.

In several embodiments of the invention, methods for the production ofPHA are provided. In one embodiment, the method comprises: (a) providinga microorganism culture comprising PHA-containing biomass, (b) removinga portion of the PHA-containing biomass from the culture, (c) extractinga portion of the PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass, (d) returning the PHA-reducedbiomass to the culture to cause the culture to convert the carbon withinthe PHA-reduced biomass into PHA, and (e) purifying the isolated PHA.

In one embodiment, the microorganism culture utilizes the PHA-reducedbiomass, or derivatives thereof, such as carbon dioxide, methane, orvolatile organic acids, volatile fatty acids, volatile organiccompounds, non-methane organic compounds, and one or morecarbon-containing gas as a source of carbon. In one embodiment, the gasis selected from the group consisting of methane, carbon dioxide,volatile organic compounds, hydrocarbons, and combinations thereof. Inone embodiment, the gas is derived from one or more sources from thegroup consisting of: landfills, wastewater treatment plants, powerproduction facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, cement production facilities, and/oranaerobic organic waste digesters.

In some embodiments, the carbon in the PHA-reduced biomass is derivedfrom one or more gases from the group consisting of: methane, biogas,carbon dioxide, volatile organic compounds, natural gas, wastewatertreatment methane and VOCs, and hydrocarbons.

In some embodiments, natural and/or artificial light is utilized toinduce the metabolism of the carbon dioxide by the culture.

In some embodiments, the microorganism culture comprises one strain, ora consortium of strains, of microorganisms, including one or moremicroorganisms selected from the group consisting of: bacteria, fungi,yeast, and algae, and combinations thereof.

In some embodiments, the microorganism culture comprises one or moremicroorganisms from the group consisting of: methanotrophicmicroorganisms, carbon-dioxide utilizing microorganisms, anaerobicmicroorganisms, methanogenic microorganisms, acidogenic microorganisms,acetogenic microorganisms, heterotrophic microorganisms, autotrophicmicroorganisms, cyanobacteria, and biomass-utilizing microorganisms, andcombinations thereof.

In some embodiments, at least a portion of the microorganism culture isnaturally occurring. In some embodiments, at least a portion of themicroorganism culture is genetically engineered. In some embodiments,naturally occurring and genetically engineered microorganisms are bothused in the culture. As used herein, the term genetically engineeredshall be given its ordinary meaning and shall also refer tomicroorganisms which have been manipulated to contain foreign (e.g., notfrom that microorganism) genetic material and/or foreign proteins. Incertain embodiments, microorganisms are genetically manipulated toexpress one or more enzymes or enzymatic pathways useful in PHAproduction. In certain embodiments, microorganisms are geneticallymanipulated to express a marker, enzyme, or protein useful allowing theselective identification of the genetically engineered microorganisms.

In some embodiments, the microorganism culture is at least partiallymaintained under above-atmospheric pressure.

In some embodiments, the PHA-containing biomass includes one or moremicroorganism-derived materials selected from the group consisting of:intracellular, cellular, and/or extracellular material, including apolymer, amino acid, nucleic acid, carbohydrate, lipid, sugar,polyhydroxyalkanoate, chemical, and/or metabolic derivative,intermediary, and/or end-product. In some embodiments, thePHA-containing biomass includes one or more microorganism-derivedmaterials selected from the group consisting of: methane, volatileorganic compounds, carbon dioxide, and organic acids.

In one embodiment, the PHA-containing biomass contains less than about95% water, including less than about 90%, about 85%, about 80%, about75%, or about 70% water.

In some embodiments, the PHA-containing biomass is mixed with one ormore chemicals, including one or more chemicals from the groupconsisting of: methylene chloride, acetone, ethanol, methanol, ketones,alcohols, chloroform, and dichloroethane, or combinations thereof.

In one embodiment, the PHA-containing biomass is processed throughhomogenization, heat treatment, pH treatment, enzyme treatment, solventtreatment, spray drying, freeze drying, sonication, and microwavetreatment, or combinations thereof.

In one embodiment, the PHA-reduced biomass includes the PHA-containingbiomass wherein at least a portion of the PHA has been removed from thePHA-containing biomass. In another embodiment, the PHA-reduced biomassincludes methane, carbon dioxide, and organic compounds produced fromthe PHA-reduced biomass.

In some embodiments, the PHA-reduced biomass is subject to dewatering,chemical treatment, sonication, additional PHA extraction,homogenization, heat treatment, pH treatment, hypochlorite treatment,microwave treatment, microbiological treatment, including both aerobicand anaerobic digestion, solvent treatment, water washing, solventwashing, and/or drying, including simple or fractional distillation,spray drying, freeze drying, and/or oven drying, or combinationsthereof.

In several embodiments, the microorganism culture is maintained in asterile, semi-sterile, or non-sterile environment.

In one embodiment, the PHA includes one or more PHA selected from thegroup consisting of: polyhydroxybutyrate (PHB), polyhydroxyvalerate(PHV), polyhydroxybutyrate-covalerate (PHB/V), polyhydroxyhexanoate(PHHx), and short chain length (SCL), medium chain length (MCL), andlong chain length (LCL) PHAs.

In several embodiments, the metabolism, growth, reproduction, and/or PHAsynthesis of the culture is controlled, manipulated, and/or affected bya growth medium. In some embodiments, the bioavailable and/or totalconcentration of nutrients within the growth medium, such as copper,iron, oxygen, methane, carbon dioxide, nitrogen, magnesium, potassium,calcium, phosphorus, EDTA, calcium, sodium, boron, zinc, aluminum,nickel, sulfur, manganese, chlorine, chromium, molybdenum, and/orcombinations thereof are manipulated (e.g., increased, decreased, ormaintained) in order to control the metabolism, growth, reproduction,and/or PHA synthesis of the culture In some embodiments, a singlenutrient in the growth medium is manipulated, while in some embodiments,more than one nutrient in the growth medium is manipulated to achievethe desired effect on the culture. It shall be appreciated thatmanipulation of a nutrient (or other component of a culture medium) isperformed, in several embodiments, in order to maintain the overallconcentration of that nutrient in the medium over time within a certaindesired range (e.g., if a nutrient is consumed by the microorganisms, a“replacement” amount of that nutrient is added to the medium).

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is induced and/or controlled by manipulating the composition of themedium. As discussed herein, the conversion of PHA-reduced biomass intothe PHA can be controlled in a time-dependent manner to maximize theefficiency of conversion. In some embodiments, conversion to PHAproduction is induced about 1-12 hours, about 5-15 hours, or about 8-24hours after PHA-reduced biomass is re-introduced into the culture. Insome embodiments, longer times, e.g., about 24 hours to several days orweeks, are employed.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is effected by manipulating the concentration one or more elementswithin a medium selected from the group consisting of: nitrogen,methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium, iron,copper, hydrogen, hydrogen ions, hydroxyl, hydroxyl ions, sulfate,manganese, calcium, chlorine, boron, zinc, aluminum, nickel, and/orsodium, and combinations thereof.

Methods used in several embodiments disclosed herein to control theconcentration of elements within the medium include, but are not limitedto, automatic, continuous, batch, semi-batch, manual, injection, solidfeed, liquid, or other methods of inputting one or more chemical intothe medium, wherein the total and/or bioavailable concentration ofelements is increased, decreased, maintained, adjusted, or otherwisecontrolled at one or more time and/or physical chemical adjustmentpoints.

According to several embodiments, additional methods to adjust the totalor bioavailable concentration of one or more elements within a mineralmedia include, but are not limited to, the directed precipitation,chelation, de-chelation, and de-precipitation of elements. In oneembodiment, the directed precipitation or chelation of one or moreelement is utilized to reduce the total or bioavailable concentration ofone or more element within a medium and thereby i) induce or increasePHA production in a biomass and/or ii) control the metabolism ofmicroorganisms within a medium, including for the purpose of controllingthe specification and/or functionality of PHA. In one embodiment,methanobactin is produced and/or utilized to chelate copper and/or ironin order to impact the metabolism of methanotrophic microorganisms.

In several embodiments, the concentration of one or more elements withina growth culture medium is increased, controlled, manipulated, orotherwise managed to induce or increase the rate of PHA production inbiomass, including a microorganism culture. In one embodiment, theconcentration of phosphorus within the medium is increased to induce orincrease the rate of PHA production in a microorganism culture. In oneembodiment, the concentration of an element, e.g., phosphorus, carbondioxide, iron, copper, oxygen, methane, hydroxyl ions, hydrogen ions,and/or magnesium, within the medium is manipulated or increased to causea metabolic shift in the microorganism culture, such that the productionof non-PHA materials by the culture using carbon and/or nitrogen sources(e.g., nitrate, ammonia, ammonium, dinitrogen, urea, or amino acids) isreduced, inhibited, or otherwise impacted to enhance PHA production. Inone embodiment, the concentration of phosphorus within the medium ismanipulated or increased to reduce the utilization of nutrients,including nitrogen, oxygen, and/or carbon, for the production of non-PHAmaterials by the culture. In one embodiment, the concentration ofphosphorus within the medium is manipulated or increased to reduce theutilization of nutrients for the production of non-PHA materials by theculture and induce or increase the rate of PHA production in theculture.

In several embodiments, an increase in the concentration of phosphoruscauses a metabolic shift that favors the production of PHA at theexpense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars,methanobactin, lipids, particularly but not necessarily undergrowth-limiting conditions, including nitrogen (e.g., nitrate, ammonia,ammonium, dinitrogen, urea, or amino acids), oxygen, magnesium,potassium, iron, copper, or other nutrient-limiting conditions.

In several embodiments, depending on the strain of microorganism, anincrease in the concentration of phosphorus above about 0.00 ppm, 0.01ppm, 0.02 ppm, 0.05 ppm, 0.10 ppm, 0.20 ppm, 0.50 ppm, 1.00 ppm, 1.25ppm, 1.50 ppm, 1.75 ppm, 2.00 ppm, 2.20 ppm, 2.40 ppm, 3 ppm, 4 ppm 5ppm, 6 ppm, 8 ppm, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90mM, 100 mM, 110 mM, 120 mM, 140 mM, 200 mM, 400 mM, 600 mM, 800 mM, or1000 mM, or overlapping ranges between these concentrations, causes areduction in the utilization of carbon and/or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,nucleic acids, lipids, pigments, polysaccharides, methanobactin, and/orcarbon dioxide, and, under growth-limiting conditions, an increase inthe utilization of carbon sources for the production of PHA material, asa result of metabolic changes in the culture and/or chemicalinteractions between chemicals within the media and/or culture inducedby augmented concentrations of phosphorus. The elevation of phosphorusconcentrations as a method to induce or increase the rate of PHAproduction in a biomass culture is contrary to the conventional wisdomin the field, which suggests that PHA production is induced or enhancedby reducing or eliminating the concentration of elements, such as, e.g.,phosphorus in the mineral medium. The addition or controlled elevationof an element, such as, e.g., phosphorus to a biomass system to induceor increase PHA production produces an unexpected and surprisingincrease in PHA production in a biomass system. An element such as,e.g., phosphorus may be added to the mineral media using a variety ofphosphorus sources, including phosphorus, phosphate, phosphoric acid,sodium phosphate, disodium phosphate, monosodium phosphate, and/orpotassium phosphate, dissolved carbon dioxide, Fe(II) iron, Fe(III)iron, copper sulfate, Fe-EDTA, dissolved oxygen, dissolved methane,and/or magnesium sulfate, among other potential sources. In oneembodiment, copper concentrations are manipulated or maintained above aminimum concentration, for at least a period of time, in order to reducethe concentration of methanobactin in the medium in order to increasethe purity of PHA produced by and/or extraction from a methanotrophicmicroorganism culture.

In several embodiments of the invention, the concentrations of dissolvedgases, such as methane, oxygen, carbon dioxide, and/or nitrogen, aremanipulated to increase the rate of PHA production relative to the rateof cellular production of non-PHA materials and, specifically, to causea reduction in the utilization of carbon or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,enzymes, nucleic acids, lipids, pigments, polysaccharides,methanobactin, and/or carbon dioxide, and, under some conditions,including growth-limiting conditions, further cause an increase in theutilization of carbon sources for the production of PHA material, as aresult of metabolic changes in the culture and/or chemical interactionsbetween chemicals within the media and/or culture induced by augmentedconcentrations of one or more of such dissolved gases. In oneembodiment, the concentration of methane or dissolved methane ismanipulated to above at least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1.0ppm, 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm,50 ppm, 100 ppm, 200 ppm, 300 ppm, 500 ppm, or 1000 ppm, or overlappingranges between these concentrations to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Insome embodiments, the concentration of oxygen or dissolved oxygen ismanipulated to above at least 0.0001 ppm, 0.01 ppm, 0.05 ppm, 0.1, 0.2ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1.0ppm, 1.1 ppm, 1.2 ppm, 1.3 ppm, 1.4 ppm 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5 ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm,500 ppm, 750 ppm, or 1000 ppm to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inone embodiment, the concentration of carbon dioxide or dissolved carbondioxide is manipulated to at least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm,1.0 ppm, 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm,4.5 ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30ppm, 50 ppm, 100 ppm, 200 ppm, 500 ppm, 1000 ppm, 1500 ppm, 2000 ppm,3000 ppm, 5000 ppm, 10,000 ppm, 20,000 ppm, or overlapping ranges ofthese concentrations to reduce the production of non-PHA materialsrelative to the production of PHA materials in a culture. In someembodiments, the concentration of nitrogen or dissolved nitrogen ismanipulated to above at least 0.01 ppm, 0.05 ppm, 0.1 ppm, 0.5 ppm, 1.0ppm, 1.5 ppm, 1.75 ppm, 2.0 ppm, 2.5 ppm, 3.0 ppm, 3.5 ppm, 4.0 ppm, 4.5ppm, 5.0 ppm, 6.0 ppm, 7.0 ppm, 8.0 ppm, 10 ppm, 15 ppm, 20 ppm, 30 ppm,or 50 ppm or ranges between these concentrations to reduce theproduction of non-PHA materials relative to the production of PHAmaterials in a culture. In several embodiments, an increase in theconcentration of methane, oxygen, carbon dioxide, and/or nitrogen causesa metabolic shift that favors the production of PHA at the expense ofother non-PHA materials, including a reduction in the production ofprotein, nucleic acids, polysaccharides, sugars, and/or lipids,particularly, but not necessarily, under growth-limiting, that is, PHAsynthesis, conditions.

In some embodiments, the PHA is at least partially removed from thePHA-containing biomass or otherwise purified using one or moreextraction agents or mechanisms selected from the group consisting of:methylene chloride, acetone, ethanol, methanol, dichloroethane,supercritical carbon dioxide, sonication, homogenization, water, heat,distillation, spray drying, freeze drying, centrifugation, filtration,enzymes, polymers, surfactants, co-solvents, hydrolyzers, acids, bases,hypochlorite, peroxides, bleaches, ozone, EDTA, miscible agents, and/orcombinations thereof.

In one embodiment, the extraction process is substantially carried outat intracellular temperatures less than 100° C. In other embodiments,temperatures for extraction range from about 10° C. to 30° C., fromabout 30° C. to 50° C., from about 50° C. to 70° C., from about 70° C.to 90° C., from about 90° C. to about 120° C., from about 100° C. toabout 140° C., from about 20° C. to 150° C., or from about 120° C. toabout 200° C., or higher. In another embodiment, cells are reused forpolymerization following the extraction process as viable cells.

In some embodiments, PHA-containing biomass is treated with one or morechemical treatment steps to control, modify, or increase theconcentration or functional characteristics (e.g., molecular weight,monomer composition, melt flow profile, purity, non-PHA residualsconcentration, protein concentration, DNA concentration, antibodyconcentration, antioxidant concentration) of PHA in a PHA-containingmaterial or biomass.

As used herein, the terms “functional properties” and “functionalcharacteristics” shall be given their ordinary meanings and shall alsorefer to the specification, features, qualities, traits, or attributesof PHA. The functional characteristics of the generated PHA include, butare not limited to molecular weight, polydispersity and/orpolydispersity index, melt flow and/or melt index, monomer composition,co-polymer structure, melt index, non-PHA material concentration,purity, impact strength, density, specific viscosity, viscosityresistance, acid resistance, mechanical shear strength, flexularmodulus, elongation at break, freeze-thaw stability, processingconditions tolerance, shelf-life/stability, hygroscopicity, and color.As used herein, the term “polydispersity index” (or PDI), shall be givenits ordinary meaning and shall be considered a measure of thedistribution of molecular mass of a given polymer sample (calculated asthe weight average molecular weight divided by the number averagemolecular weight). Advantageously, several embodiments of the processesdisclosed herein may be carried out in sterile, semi-sterile, ornon-sterile conditions. In several embodiments, consistency in more thanone of these functional properties is achieved. For example, in someembodiments, consistent molecular weight, polydispersity, andcombinations thereof are achieved. In one embodiment, temperature isused to control, modify, reduce, or optimize the molecular weight,polydispersity, melt flow, and other characteristics of PHA. In oneembodiment, temperature and/or time is used to control the molecularweight of PHA between the range of about 5,000,000 and about 10,000Daltons. In several embodiments the molecular weight of PHA rangesbetween about 5,000,000 and about 2,500,000 Daltons, between about2,500,000 and about 1,000,000 Daltons, between about 1,000,000 and about750,000 Daltons, between about 750,000 and about 500,000 Daltons,between about 500,000 and about 250,000 Daltons, between about 250,000and about 100,000 Daltons, between about 100,000 and about 50,000Daltons, between about 50,000 and about 10,000 Daltons, and overlappingranges thereof. In one embodiment, a slurry comprising PHA-containingbiomass and a culture media is subject to one or more water removalsteps or water addition steps to increase the concentration of PHA in aPHA-containing biomass. In one embodiment, the water removal step is adewatering step or combination of dewatering steps, such ascentrifugation, filtration, spray drying, flash drying, and/or chemicaldewatering (e.g., with acetone, ethanol, or methanol, or combinationsthereof), wherein at least a portion of the water concentration relativeto the concentration of PHA-containing biomass in the slurry is reduced.In one embodiment, a temperature and/or pressure control step is carriedout under atmospheric (0 psi), sub-atmospheric (−100-0 psi), orabove-atmospheric pressure (e.g., 0-30,000 psi) and at temperatureconditions wherein the PHA-containing biomass, or the liquid in and/oraround the PHA-containing biomass, is maintained, for at least a periodof time, at a temperature ranging from −30 to 10 degrees Celsius, 10degrees Celsius to 100 degrees Celsius, 10 degrees Celsius to 150degrees Celsius, 20 degrees Celsius to 250 degrees Celsius, and/or 100to 200 degrees Celsius, including overlapping ranges thereof. In oneembodiment, the PHA-containing biomass is subject to a dewatering stepbefore or after the temperature and/or pressure control step, whereinthe dewatering step is centrifugation, filtration, and/or spray drying,to produce a fully or partially de-watered PHA-containing biomass orPHA-containing biomass slurry, wherein the water concentration of thedried slurry is less than 99%, 95%, 80%, 60%, 40%, 30%, 20%, 10%, 5%,3%, 2%, or 1% water. In one embodiment, the PHA-containing biomass issubject to a temperature control step, wherein the water or liquidchemicals within and/or around the biomass is controlled and maintainedat a temperature of at least −30, −10, −5, −4, −3, −2, −1, 0, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 degreesCelsius or ranges between those temperatures. In one embodiment, thePHA-biomass is not dried prior to such temperature control step. In oneembodiment, the PHA-containing biomass is dried or de-watered prior tosuch temperature control step. In one embodiment, the PHA-containingbiomass is filtered or centrifuged following the temperature controlstep. In one embodiment, the PHA-containing cell slurry is notdewatered, for example, by centrifugation or other drying mechanism,prior to the temperature control step. In one embodiment, a mechanism toimpart shear onto or into the PHA-containing biomass is coupled with atemperature control step; such shear may be imparted in the form of oneor more shear induction mechanisms including, but not limited to e.g., acentrifugal pump, agitator, blender, high shear mixer, vortex mixer,etc. In one embodiment, the PHA-containing biomass is dewatered in onestep and the treated PHA-containing biomass is further dewatered in oneor more additional steps. In one embodiment, the PHA-containing biomassis dewatered, water and/or other chemicals are added and temperatureand/or pressure is controlled, and the treated PHA-containing biomass isfurther dewatered and/or purified. In one embodiment, the water and/orchemicals within, around, and/or added to the PHA-containing biomass istemperature and/or pressure controlled, and the treated PHA-containingbiomass is further purified in one or more purification steps. In oneembodiment, the temperature control step process time is approximately 1second, 5 seconds, 10 seconds, 25 seconds, 60 seconds, 2 minutes, 5minutes, 20 minutes, 45 minutes, 1 hour, 2 hours, 5 hours, 6 hours, 7hours, 12 hours, 15 hours, 24 hours, 36 hours, or 48 hours, or rangesbetween those times. In one embodiment, inorganic materials may be usedto effect PHA modification, purification, or extraction, includingcarbon dioxide and dinitrogen. In one embodiment, the PHA-containingslurry or biomass is treated with carbon dioxide under elevatedtemperatures and pressures, including supercritical ranges, to inducePHA extraction or functional modification of PHA. In one embodiment,solvents, including methylene chloride, enzymes, super critical fluids,acetone, chloroform, dichloroethane, ethanol, plasticizers, acids,bases, polymers, and/or methanol are used, alone or in combination, inconjunction with any of the above steps to improve efficiency, includingincreasing purity, recovery, extractability, solubility, reaction speed,odor, color, recyclability, biodegradability, toxicity, endotoxinremoval rate, and the like. In one embodiment, solvents or extractionmaterials may be used or recycled for biomass production, biogasproduction, and/or PHA synthesis.

In one embodiment, chemicals are added to a PHA-containing biomass tocause the crystallization of PHA. In one embodiment, methylene chloride,carbon dioxide, acetone, water, dichloroethane, or methanol may be addedto a PHA-containing biomass in order to induce the crystallization ofPHA in the PHA-containing biomass. In some embodiments, this step may beuseful for the downstream processing of PHA, wherein crystallized PHA isless prone than amorphous PHA to degradation, including molecular weightloss, when contacted with extraction chemicals, including solvents,enzymes, acids, bases, and bleach. In one embodiment, silicon, silica,derivatives thereof, and/or chemicals containing silicon may be added tothe PHA-containing biomass in order to impact the metabolic status ofthe culture, and thereby control the functional characteristics of thePHA produced by the culture, including one or more of the following:molecular weight, monomer composition, co-polymer structure, melt index,and polydispersity.

In one embodiment, the removal of the PHA from the culture causes theculture to be temporarily deactivated, such that the culture, orelements thereof, may be further used for the synthesis of PHA. Incertain embodiments, deactivation is beneficial because it allows forthe delay of PHA production, transfer of material to another productionarea, and the like. In some embodiments, deactivation allows a tailoredPHA production time frame. In some embodiments, the reuse of cells forpolymerization is beneficial because it avoids or reduces the need toproduce new biomass prior to polymerization, thereby reducing thecarbon, chemical, and energy requirement of PHA production.

In one embodiment, a PHA produced according to the several embodimentsdescribed herein is provided.

In several embodiments, processes for the production of PHA from acarbon-containing gas are provided. In one embodiment, the processcomprises the steps of: a) providing a growth medium comprising amicroorganism culture capable of utilizing the carbon within one or morecarbon-containing gas and PHA-reduced biomass, b) manipulating themedium to cause the culture to produce PHA, c) removing at least aportion of the PHA within the culture to create substantially isolatedPHA and substantially PHA-reduced biomass, d) purifying the isolatedPHA, and e) returning the PHA-reduced biomass to the culture to causethe culture to metabolize the carbon within the PHA-reduced biomass intoPHA.

In one embodiment, the carbon-containing gas is selected from the groupconsisting of: methane, carbon dioxide, toluene, xylene, butane, ethane,methylene chloride, acetone, ethanol, propane, methanol, vinyl chloride,volatile organic compounds, hydrocarbons, and combinations thereof. Asdiscussed herein, in some embodiments, non-gaseous carbon-containingmaterial can be used, at least in part, for the production of PHA.

In one embodiment, the invention comprises manipulating theconcentration of elements, e.g., copper, iron, phosphorus, oxygen,methane, carbon dioxide, in the culture medium to control theconcentration of sMMO and/or pMMO produced by a methanotrophic culturein order to control the relative ratio of sMMO to pMMO in the culture,including over time and over multiple growth and polymerization cycles,and thereby control one or more of the growth conditions, metabolicstatus, metabolic disposition, and/or specification of PHA (e.g.,molecular weight, polydispersity, melt flow, monomer composition, etc.)produced by the culture, including over time. In some embodiments, sMMOis expressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingsMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout 100%, and overlapping ranges thereof. Simultaneously, orindependently, in some embodiments, pMMO is expressed in a range betweenabout 0% and 100% of a methanotrophic culture by dry cell weight, as apercentage of microorganisms expressing pMMO, or as a percentage oftotal MMO expressed by one or more methanotrophic cells, includingbetween 0% and 1%, between about 1% and about 2%, between about 2% andabout 3%, between about 3% and about 5%, between about 5% and about 10%,between about 10% and about 20%, between about 20% and about 30%,between about 30% and about 50%, between about 50% and about 70%,between about 70% and about 80%, between about 80% and about 90%,between about 90% and about 95%, between about 95% and about 100%, andoverlapping ranges thereof. In some embodiments, the ratio of sMMO topMMO produced in a methanotrophic culture is controlled to control thespecification of PHA produced by a culture. In some embodiments, therelative weight ratio of sMMO to pMMO in a methanotrophic culture is atleast or approximately 0 to 1, approximately 0.0000001 to 1,approximately 0.0001 to 1, approximately 0.001 to 1, approximately 0.01to 1, approximately 0.1 to 1, approximately 1 to 1, approximately 2 to1, approximately 3 to 1, approximately 5 to 1, approximately 10 to 1,approximately 15 to 1, approximately 20 to 1, approximately 25 to 1,approximately 30 to 1, approximately 35 to 1, approximately 50 to 1,approximately 65 to 1, approximately 70 to 1, approximately 80 to 1,approximately 90 to 1, approximately 95 to 1, approximately 98 to 1,approximately 99 to 1, approximately 100 to 1, approximately 1000 to 1,approximately 10,000 to 1, approximately 100,000 to 1, or approximately1,000,000 to 1, respectively. In some embodiments, the relative weightratio of pMMO to sMMO in a methanotrophic culture is approximately 0 to1, approximately 0.0000001 to 1, approximately 0.0001 to 1,approximately 0.001 to 1, approximately 0.01 to 1, approximately 0.1 to1, approximately 1 to 1, approximately 2 to 1, approximately 3 to 1,approximately 5 to 1, approximately 10 to 1, approximately 15 to 1,approximately 20 to 1, approximately 25 to 1, approximately 30 to 1,approximately 35 to 1, approximately 50 to 1, approximately 65 to 1,approximately 70 to 1, approximately 80 to 1, approximately 90 to 1,approximately 95 to 1, approximately 98 to 1, approximately 99 to 1,approximately 100 to 1, approximately 1000 to 1, approximately 10,000 to1, approximately 100,000 to 1, or approximately 1,000,000 to 1.

In some embodiments, by controlling the relative concentrations of sMMOand pMMO produced by a culture of methanotrophic microorganisms, it ispossible to control the metabolic status of the culture and therebycontrol the type (and characteristics) of PHA and other cellularmaterial produced by the culture, particularly in the presence of one ormore of the following: volatile organic compounds, fatty acids, volatilefatty acids, methanol, formate, acetone, acetate, acetic acid, formicacid, dissolved carbon dioxide, dissolved methane, dissolved oxygen,carbon-containing materials, ammonia, ammonium, and other elements orcompounds that impact the metabolism of a culture of methanotrophicmicroorganisms in a certain manner according to the relativeconcentration of sMMO or pMMO in such a culture. In some embodiments,sMMO and/or pMMO is expressed in a range between about 0% and 100% of amethanotrophic culture by dry cell weight, as a percentage ofmicroorganisms expressing sMMO or pMMO, or as a percentage of total MMOexpressed by one or more methanotrophic cells, including between 0% and1%, between about 1% and about 2%, between about 2% and about 3%,between about 3% and about 5%, between about 5% and about 10%, betweenabout 10% and about 20%, between about 20% and about 30%, between about30% and about 50%, between about 50% and about 70%, between about 70%and about 80%, between about 80% and about 90%, between about 90% andabout 95%, between about 95% and about or 100%, and overlapping rangesthereof prior to, during, throughout, or after a PHA production phase.

In one embodiment, sMMO is not expressed, or is expressed in low amounts(e.g., less than about 35%, about 25%, about 15%, about 5%, about 3% orabout 1%), in a methanotrophic culture prior to, during, throughout, orafter a PHA production phase. In some embodiments, the directed orcontrolled absence or reduction of sMMO in a methanotrophic cultureproducing PHA, particularly in the presence of non-methane organiccompounds that can be metabolized by methanotrophic microorganisms,engenders PHA production stability, consistency, and control byselectively shielding against the metabolism of one or some or manynon-methane organic compounds that might otherwise be metabolized in thepresence of sMMO, which enables the metabolism of a larger group ofnon-methane compounds than pMMO. Similarly, in one embodiment, pMMO isnot expressed, or is expressed in low amounts (e.g., less than about35%, about 25%, about 15%, about 5%, about 3% or about 1%), in amethanotrophic culture prior to, during, throughout, or after a PHAproduction phase. In some embodiments, the directed or controlledabsence or reduction of pMMO in a methanotrophic culture producing PHA,particularly in the presence of non-methane organic compounds that canbe metabolized by methanotrophic microorganisms, engenders PHAproduction stability, consistency, and control by selectively inducingor promoting the metabolism of one or some or many non-methane organiccompounds that might otherwise be metabolized using pMMO. Further, insome methanotrophic cultures, sMMO promotes PHA synthesis at highintracellular concentrations by reducing cellular production of non-PHAmaterials, particularly as compared to PHA synthesis using pMMO. Bycontrolling the concentration of sMMO relative to pMMO in amethanotrophic microorganism culture in the presence of methane and/ornon-methane organic compounds, including VOCs, volatile fatty acids,acetone, formate, ethane, propane, it is possible to control thespecification or type of PHA produced by the culture, including themolecular weight, polydispersity, and other similar functionalcharacteristics as disclosed herein. In some embodiments, it ispreferable to maintain the concentration of copper in the culture mediain order to promote sMMO production. In some embodiments, the productionof sMMO in many, most, or substantially all of the methanotrophic cellsenables the culture to produce more PHA when subject to a nutrientlimiting step than would otherwise be produced if the relative ratio ofpMMO in the culture was higher prior to the nutrient limiting step. Insome embodiments, it is preferable to maintain the concentration ofcopper in the culture media in order to promote pMMO production. In someembodiments, the production of pMMO in many, most, or substantially allof the methanotrophic cells enables the culture to produce more PHA whensubject to a nutrient limiting step than would otherwise be produced ifthe relative ratio of sMMO in the culture was higher prior to thenutrient limiting step. In one embodiment, one or more methanotrophiccells or cultures are subject to repeated growth and PHA synthesiscycles or steps, wherein the relative concentration of sMMO to pMMO inthe cells or cultures is controlled or caused to remain approximatelysimilar or substantially the same (e.g., within about 5% to about 10%,with about 10% to about 20%, within about 20% to about 30%, within about30% to about 40%, within about 40% to about 50%, within about 50 toabout 75%) or the same in each new cycle or step in order to control orkeep substantially the same (e.g., within about 5% to about 10%, withabout 10% to about 20%, within about 20% to about 30%, within about 30%to about 40%, within about 40% to about 50%, within about 50% to about75% across production runs) the specification, characteristics, and/orfunctionality (e.g., molecular weight, monomer composition, melt flowindex, polydispersity, non-PHA material concentration, and/or purity) ofthe PHA produced by or extractable from the culture or cultures in eachnew or repetitive cycle with the same or new cells.

In one embodiment, the invention comprises a PHA comprising carbonderived from PHA-reduced biomass, wherein the PHA-reduced biomasscomprises carbon derived from one or more carbon-containing gassesand/or one or more additional sources of carbon (either gaseous ornon-gaseous). In one embodiment, PHA is co-mingled and/or melted withPHA-reduced biomass to improve the functional characteristics of thePHA. In one embodiment, PHA is co-mixed and/or melted with PHA-removedbiomass and/or microorganism biomass to improve the functionalcharacteristics of PHA. In one embodiment, the percentage of non-PHAmicroorganism biomass included in a PHA, PHA compound, or PHA mixture isabout 0.00001% to about 0.001%, about 0.001% to about 0.01%, about 0.01%to about 0.1%, to about 0.1% to about 0.5%, about 0.5% to about 1%,about 1%, to about 2%, about 2% to about 3%, about 3% to about 5%, about5% to about 7%, about 7% to about 10%, about 10% to about 15%, about 15%to about 20%, about 20% to about 30%, about 30% to about 40%, about 40%to about 50%, about 50% to about 60%, about 60% to about 70%, about 70%to about 80%, about 80% to about 90%, about 90% to about 98%, about 98%to about 99.99%, and overlapping ranges thereof. In some embodiments,the inclusion of microorganism biomass to a PHA improves the functionalcharacteristics of a PHA by acting as one or more of the following:nucleating agent, plasticizer, compatibilizer, melt flow modifier, moldrelease agent, filler, strength modifier, elasticity modifier, ordensity modifier. In some embodiments, the microscopic size ofmicroorganism biomass, including nucleic acids and proteins, isparticularly and surprisingly effective as a functionalization agent forPHA. In some embodiments, microorganism biomass acts as a surprisinglyeffective compatibilizer for PHA and non-PHA polymers, such aspolypropylene and polyethylene. In one embodiment, the biomass issubject to a processing step in order to make the biomass, or at least aportion of the biomass, miscible with the PHA and/or non-PHA polymer,which yields unexpected and surprising functional improvement of thebiomass as a blend component. Such processing step may include: heat,shear, pressure, solvent extraction, washing, filtration,centrifugation, sonication, enzymatic treatment, super critical materialtreatment, cellular dissolution, flocculation, acid and/or basetreatment, drying, lysing, and/or chemical treatment, wherein saidchemicals may include solvents, cell dissolution agents, cellmetabolizing agents, polymers, plasticizers, compatibilization agents,nucleating agents, including processing steps that enable PHA containedin said biomass to become miscible with said biomass and/or othermaterials, including a second polymer or carrier agent. In oneembodiment, microorganism biomass is mixed with a PHA to modify one ormore of the nucleation, plasticization, compatibilization, melt flow,density, strength, elongation, elasticity, mold strength, mold release,and/or bulk density characteristics of a PHA, which may be melted,extruded, film blown, die cast, pressed, injection molded, or otherwiseprocessed. In one embodiment, PHA is partially or not removed fromPHA-containing microorganism biomass prior to melt processing, e.g.,extrusion, injection molding, etc. In one embodiment, non-PHA biomassparts or materials are caused to remain with PHA derived from aPHA-containing biomass in order to modify or control the functionalcharacteristics of a PHA material. In one embodiment, PHA is present inPHA-containing biomass at a concentration ranging from 1-99.99999%,1-99.99%, 1-99%, 5-99%, 10-99%, 20-99%, 30-99%, 50-99%, 70-99%, 80-99%,90-99%, 95-99%, 98-99%, and overlapping ranges thereof. In oneembodiment, PHA, microorganism biomass, and/or one or more non-PHAmaterial, polymer, or thermoplastic are mixed, melted, or processedtogether. In one embodiment, the non-PHA polymer or material consists ofone or more of the following solvents, cell dissolution agents, cellmetabolizing agents, polymers, plasticizers, compatibilization agents,miscible agents, and nucleating agents: polypropylene, polyethylene,polystyrene, polycarbonate, Acrylonitrile butadiene styrene,Polyethylene terephthalate, Polyvinyl chloride, Fluoropolymers, LiquidCrystal Polymers, Acrylic, Polyamide/imide, Polyarylate, Acetal,Polyetherimide, Polyetherketone, Nylon, Polyphenylene Sulfide,Polysulfone, Cellulosics, Polyester, Polyurethane, Polyphenylene oxide,Polyphenylene Ether, Styrene Acrylonitrile, Styrene Maleic Anhydride,Thermoplastic Elastomer, Ultra High Molecular Weight Polyethylene,Epoxy, Melamine Molding Compound, Phenolic, Unsaturated Polyester,Polyurethane Isocyanates, Urea Molding Compound, Vinyl Ester,Polyetheretherketone, Polyoxymethylene plastic, Polyphenylene sulfide,Polyetherketone, Polysulphone, Polybutylene terephthalate, Polyacrylicacid, Cross-linked polyethylene, Polyimide, Ethylene vinyl acetate,Polyvinyl chloride, Polyvinyl acetate, Polyvinyl acetateco-Polyvinylpyrrolidone, Polyvinylpyrrolidone, Polyvinyl alcohol,Cellulose, Lignin, Cellulose Acetate Butryate, Polypropylene,Polypropylene Carbonate, Propylene Carbonate, Polyethylene, ethylalcohol, ethylene glycol, ethylene carbonate, glycerol, polyethyleneglycol, pentaerythritol, polyadipate, dioctyl adipate, triacetylglycerol, triacetyl glycerol-co-polyadipate, tributyrin, triacetin,chitosan, polyglycidyl methacrylate, polyglycidyl metahcrylate,oxypropylated glycerine, polyethylene oxide, lauric acid, trilaurin,citrate esters, triethyl citrate, tributyl citrate, acetyl tri-n-hexylcitrate, saccharin, boron nitride, thymine, melamine, ammonium chloride,talc, lanthanum oxide, terbium oxide, cyclodextrin, organophosphoruscompounds, sorbitol, sorbitol acetal, sodium benzoate, clay, calciumcarbonate, sodium chloride, titanium dioxide, metal phosphate, glycerolmonostearate, glycerol tristearate, 1,2-hydroxystearate, celluloseacetate propionate, polyepichlorohydrin, polyvinylphenol, polymethylmethacrylate, polyvinylidene fluoride, polymethyl acrylate,polyepichlorohydrin-co-ethylene oxide, polyvinyl idenechloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, Styrene maleic anhydride,Styrene-acrylonitrile, Poly(methyl methacrylate),Polytetrafluoroethylene, Polybutylene. Polylactic acid, Polyvinylidenechloride, and/or other similar materials or combinations of thesematerials, including mold release agents, plasticizers, solvents,solvent-grafted polymer, salts, nucleating agents, cross-linking agents,filaments, water, antioxidants, compatibilizers, co-polymers, peroxides,alcohols, ketones, polyolefins, chlorinated solvents, non-chlorinatedsolvents, aliphatic hydrocarbons, hydrophilic agents, hydrophobicagents, enzymes, PHA miscible agents, pigments, stabilizers, and/orrubbers. In several embodiments, the additional of one or more of suchnon-PHA polymer or material advantageously improves the post-productionhandling of PHA. In one embodiment, PHA, methanotrophic, autotrophic,and/or heterotrophic microorganism biomass, and a non-PHA polymer aremixed and melted together. In one embodiment, the concentration ofnon-PHA microorganism biomass in such a mixture ranges from 0.0001% to90%, 0.1% to 30%, 0.1% to 10%, or 0.5% to 8%, and overlapping rangesthereof. In one embodiment, the concentration of methanobactin in PHA iscontrolled to modify the functional characteristics of the PHA,including color, odor, brittleness, flexibility, antioxidant activity,antiviral activity, and/or antibacterial activity. In one embodiment,reducing the concentration of methanobactin in the PHA reduces the brownor yellow shade of the PHA and increases the flexibility of the PHA. Inanother embodiment, increasing the concentration of methanobactin in thePHA increases the antimicrobial (e.g., antibacterial, antiviral) and/orantioxidant activity or potential activity or the PHA. In oneembodiment, a method is provided for improving the functionalcharacteristics of a PHA polymer through the melting and cooling of thePHA polymer in the presence of a dual-miscible biomass agent and asecond polymer, comprising the steps of: (a) providing a first polymer,a biomass, and a second polymer, wherein the first polymer is a PHA, (b)subjecting the biomass to a processing step comprising heat, pressure,solvent washing, filtration, centrifugation, super critical solventextraction, and/or shear, wherein the processing step renders at least aportion of the biomass miscible with the first polymer and the secondpolymer, (c) contacting the first polymer with the biomass and thesecond polymer to form a compound, (d) heating the compound to between50 degrees Celsius and 250 degrees Celsius, and (d) causing the biomassto effect a functional modification of the first polymer, the secondpolymer, and the combination of the first polymer and second polymer,wherein the functional modification comprises plasticization,nucleation, compatibilization, melt flow modification, strengthening,and/or elasticization.

In one embodiment, a PHA derived from a carbon-containing greenhousegas, or greenhouse gas emission including methane, carbon dioxide, orcombinations thereof, is provided. In some embodiments, use of such agas is particularly advantageous, as it allows for the simultaneousproduction of PHA at lower energy costs and higher efficiencies, butalso removes a portion of a destructive gas from the atmosphere, orprevents a destructive gas from entering the atmosphere. In someembodiments, processes and systems as disclosed herein are particularlywell suited for use near sources of such gases (e.g., landfills, powerproduction plants, anaerobic digesters, etc.) for onsite conversion ofharmful gasses to a commercially valuable product.

In several embodiments, processes for the oxidation of methane areprovided. In one embodiment, the process comprises: providing a cultureof methanotrophic and autotrophic microorganisms, providing a growthculture medium comprising dissolved methane and carbon dioxide, andcontacting the culture with light to cause the culture to convert thecarbon dioxide into oxygen, whereby the culture utilizes the oxygen tooxidize the methane, thereby reducing or eliminating the need for anextraneous source of oxygen to drive methanotrophic metabolism.

In some embodiments, the light used to contact the culture is artificiallight. In some embodiments, the light used to contact the culture isnatural light. In other embodiments, combinations of natural andartificial light are used. In some such embodiments, wavelengths ofartificial light are specifically filtered out or controlled such thatthe culture is exposed to a broader or more controlled overall spectrumof light (e.g., the sum of wavelengths of natural light and artificiallight). In some embodiments, the source of light also functions togenerate heat, which can be used to maintain optimal culturetemperatures. In other embodiments, light input is regulated by time,such that specific cultures of autotrophic and/or heterotrophicmicroorganisms are selected for or optimized according to the durationand/or pattern of light injection (e.g., 0-12 or 12-24 hours lightinjection, 0-12 or 12-24 hours dark incubation, multi-second pulsation,etc.).

In one embodiment, the addition of light reduces the need for exogenousoxygen sources. While such embodiments provide an advantage in reducingcosts of input materials, in some embodiments, an exogenous source ofoxygen, including air, is added to the culture.

In some embodiments, the addition of autotrophic microorganisms to theculture impacts the metabolism of the culture. In such an embodiment,the timed and planned addition, activation, or metabolic enhancement ofautotrophic organisms can be based on the desire for changing the rateof methane oxidation.

In some embodiments, a system is used for PHA production comprisingproviding i) a culture of autotrophic, methanotrophic, methanogenic,and/or heterotrophic microorganisms and ii) a first gas comprisingcarbon dioxide, methane, volatile organic compounds, oxygen, and/orother gas, whereby the culture of microorganisms are caused to used thefirst gas to generate a second gas comprising carbon dioxide, methane,oxygen, volatile organic compounds, and/or other gas, whereby theculture subsequently is caused to utilize the second gas for thegeneration of PHA, which can then be isolated and purified according toseveral embodiments disclosed herein. In some embodiments, the first gascan be methane, carbon dioxide, oxygen, or volatile organic compounds.In other embodiments, the second gas can be oxygen, methane, carbondioxide, or other volatile organic compounds. In some embodiments,microorganisms can be used to convert carbon dioxide to biomass whichcan in turn be used to produce methane, which can be subsequently usedto produce PHA. In other embodiments, microorganisms can be used toconvert carbon dioxide to oxygen which can in turn be used to producePHA. As disclosed herein, the products generated at each of these stepsmay be recycled (e.g., splitting a portion of the autotrophic cultureand recycling it to generate additional biomass, generating reduced-PHAbiomass and recycling it into the methanotrophic culture to generateadditional biomass and additional PHA).

In several embodiments, processes for producing autotrophicmicroorganisms using only methane as a carbon input are provided. In oneembodiment, the process comprises: adding methane, oxygen, andmethane-utilizing microorganisms to a culture of autotrophicmicroorganisms, whereby the methane-utilizing microorganisms convert themethane into carbon dioxide, and whereby the autotrophic microorganismsutilize the carbon dioxide as a source of carbon, thereby reducing oreliminating the need for an extraneous source of carbon dioxide to driveautotrophic metabolism. In some embodiments, addition of methanotrophicand/or heterotrophic microorganisms to the culture impacts themetabolism of the autotrophic microorganisms. As discussed herein, thepurposeful addition of such microorganisms at particular times allowsfor specific levels of control over the overall output and operation ofthe system.

In several embodiments, processes for oxidizing methane at lowconcentrations are provided. In one embodiment, the process comprises:culturing methanotrophic microorganisms in a medium comprising water,dissolved methane, dissolved oxygen, and mineral salts, adding methanolto the medium at a rate and volume sufficient to cause themicroorganisms to reduce the concentration of the methane in the medium,whereby substantially all of the methane within the medium is utilized,thereby enabling methanotrophic microorganisms to metabolize methanepresent at low bioavailable concentrations. In some embodiments, gascontaining less than 20% methane by volume is contacted with the medium.In some embodiments, gas containing less than 1% methane by volume iscontacted with the medium. In some embodiments, the methanol is producedby microorganism metabolism.

In several embodiments, processes for separating water frommicroorganism biomass are provided. In one embodiment, the processcomprises: providing biomass mixed with water in a liquid medium, mixingthe medium with a liquid agent selected from the group consisting ofketones, alcohols, chlorinated solvents, derivatives thereof, orcombinations thereof, and subjecting the mixture to a filtration step.In several embodiments, this enables the efficient separation of biomassfrom water. In some embodiments, such methods reduce or eliminate theneed for centrifugation in the separation process. In some embodiments,the liquid agent is acetone, ethanol, isopropanol, and/or methanol. Inother embodiments, other liquid agents that are miscible with water areused to separate the biomass from the aqueous portion of the mixture. Inseveral embodiments separation is achieved by centrifugation(high-speed, low-speed), gravity separation, multi-stage filtration, orcombinations thereof.

In several embodiments, processes for extracting a polyhydroxyalkanoatefrom a PHA-containing biomass are provided. In one embodiment, theprocess comprises the steps of: (a) providing a PHA-containing biomasscomprising PHA and water, (b) mixing said biomass with a solvent at atemperature sufficient to dissolve at least a portion of said PHA intosaid solvent and at a pressure sufficient to enable substantially all orpart of said solvent to remain in liquid phase, thereby producing aPHA-lean biomass phase and a PHA-rich solvent phase comprising water,PHA and solvent (c) separating said PHA-rich solvent phase from saidPHA-lean biomass phase at a temperature and pressure sufficient toenable substantially all or part of said solvent to remain in liquidphase and prevent substantially all or part of said PHA within saidPHA-rich solvent phase from precipitating into said water, (d) reducingthe pressure or increasing the temperature of said PHA-rich solventphase to cause said PHA-rich solvent to vaporize and said PHA toprecipitate or otherwise become a solid PHA material while maintainingthe temperature and/or pressure of the PHA-rich solvent phase to preventall or part of the temperature-dependent precipitation of said PHA intosaid water, and (e) collecting said solid PHA material, includingoptionally separating said solid PHA material from said solvent and/orsaid water.

In some embodiments, solvents include acetone, ethanol, methanol,dichloroethane, and/or methylene chloride. Depending on the solventselected, in some embodiments, separating the solid PHA material fromsolvent and/or water is achieved by increasing the temperature of themixture. In other embodiments, separation is achieved through reducingthe pressure of the solvent, PHA, and/or water. In some embodiments,combinations of temperature changes and pressure changes are used tooptimally separate solid PHA material from solvent and/or water. In someembodiments, evaporation of solvent and/or water occurs in a rapidfashion, thereby reducing the need for temperature or pressure changes.Advantageously, certain embodiments of the processes disclosed hereinmay optionally be carried out in a batch, semi-continuous, or continuousmanner. Thus, the process can be tailored to the needs of the producerat any given time.

In several embodiments, processes for modifying the functionalcharacteristics of a PHA are provided. In one embodiment, the processcomprises providing a first PHA and a second PHA, wherein the molecularweight of said second PHA is greater than the molecular weight of saidfirst PHA, and combining said first PHA with said second PHA to modifythe functional characteristics of both said first PHA and second PHA. Insome embodiments, both the first PHA and the second PHA are PHB, and insome embodiments, one or more of the first and second PHA comprisesPHBV. In one embodiment the first PHA and the second PHA is PHB or PHBV.

In some embodiments, the molecular weight of the first PHA is greaterthan about 500,000 Daltons and the molecular weight of the second PHA isless than about 500,000 Daltons. However, in some embodiments, themolecular weight can be adjusted. For example, in some embodiments, afirst PHA is subjected to a temperature sufficient to reduce themolecular weight of the first PHA. Thereafter, it can be combined withthe second PHA. It shall be appreciated that the second PHA could alsooptionally be exposed to temperature in order to adjust its molecularweight. In some embodiments, the molecular weight of the second PHA isgreater than about 800,000 Daltons. In certain embodiments, themolecular weight of said second PHA is greater than about 1,000,000Daltons. In some embodiments, the molecular weights of the first andsecond PHA are specifically tailored relative to one another, (e.g., aratio of 1:2, 1:4, 1:6, 1:8, 1:10, etc.) in order to maximize thealterations in functional characteristics.

In several embodiments, processes for increasing the penetration depthof light in a liquid are provided. In one embodiment, the processcomprises the steps of (a) directing light into a liquid medium in theform of a light path and (b) reducing the density of liquid in the lightpath.

In several embodiments, the density of the liquid in the light path isreduced by adding gas to said liquid in the light path. In someembodiments, the gas is air, oxygen, methane, carbon dioxide, nitrogen,and/or a combination thereof. In some embodiments, the gas issimultaneously added along with the light. In certain embodiments, thegas and the light are emitted or injected into the liquid through acommon material, such as a permeable or semi-permeable membrane throughwhich light and/or gas traverse. In other embodiments, the light and thegas are added separately. In such embodiments, customization of theaddition is possible. For example, gas can be added in pulses (e.g.,on/off sequences), continuously, or in bracketed time frames around theaddition of light. In some embodiments, the addition of light and gasare coordinated to maximize the penetration of the light. For example aburst of gas followed by a burst of light (or overlapping to somedegree) may advantageously increase the penetration of the light.

In several embodiments, processes for modifying the pH in amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a culture mediumcomprising water and microorganisms, (b) adding a first source ofnitrogen to the medium to cause the microorganisms to metabolize thenitrogen and thereby increase the concentration of either hydroxyl ionsor protons, respectively, in the medium, and/or (c) adding a secondsource of nitrogen to the medium to cause the microorganisms tometabolize the nitrogen and thereby increase the concentration of eitherprotons or hydroxyl ions, respectively, in the medium. In otherembodiments, a source of nitrogen is added to the culture that increasesthe pH of the medium, wherein the metabolism of the nitrogen sourcecauses the pH of the medium to decrease, thereby reducing or eliminatingthe need for an additional pH adjustment step. In one embodiment,nitrogen fixation is used to add hydroxyl ions to the culture medium,which may or may not be counterbalanced by the addition of protons fromeither biological or chemical sources. In one embodiment, nitratefixation is used to add hydroxyl ions to the culture medium, which mayor may not be counterbalanced by the addition of protons from eitherbiological or chemical sources. In one embodiment, ammonia or ammoniumfixation is used to add protons to the culture medium, which may or maynot be counterbalanced by the addition of hydroxyl ions from eitherbiological or chemical sources.

In several embodiments, low shear processes for adding gas to amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a liquid medium and a gas,(b) contacting the medium with the gas in a first container to cause atleast a portion of the gas to dissolve in the medium, (c) providing asecond container, and (d) transferring at least a portion of the liquidcomprising the gas within the first container to the second container.In some embodiments, a mixer is also provided, in order to dissolve aportion of the gas in the medium. In some embodiments, the mixer is apump or agitator or high shear mixer. In some embodiments, the mixercomprises a centrifugal pump. In still other embodiments, the gas itselfprovides a mixing function. For example, the injection of gas into amedium will result in gas bubbles, which, if released at the bottom of acontainer comprising medium, will not only promote the dissolution ofgas into the medium, but mix the medium as the bubbles rise.

In several embodiments, processes for injecting gas into a pressurizedmicroorganism culture vessel are provided. In one embodiment, theprocess comprises the steps of: providing a vessel comprising a mediumcomprising microorganisms, adding a gas into the vessel that can bemetabolized by the microorganisms, and adjusting the flow rate of thegas into the vessel according to the rate of change of pressure withinthe vessel. In some embodiments, the gas is oxygen, methane, carbondioxide, or combinations thereof. Choice of the gas depends on thevessel used and the culture within the vessel. In some embodiments,backpressure monitoring allows for optimal gas injection for a givenculture (e.g., if certain cultures react more quickly to administrationof a gas and rapidly increase pressure, flow can be coordinatelyreduced).

In one embodiment, a process for producing light in a liquid medium isprovided. In one embodiment, the process comprises the steps of: a)providing a liquid, b) providing a light-emitting unit or materialcomprising two conductive leads and a light-emitting conjuncture betweenthe conductive leads, or a material that will emit light when contactedwith electrons and c) inducing a voltage in the liquid, thereby inducingthe movement of electrons in the conductive leads of the light-emittingunit or inducing electrons to contact the material, thereby causing thelight-emitting unit or material to emit light. In some embodiments, thematerial is a phosphor, phosphoric, and/or luminescent material,including an electroluminescent phosphor.

In one embodiment, AC voltage is induced in a liquid by inserting thetwo leads of a 115V AC power source into a liquid. Without being boundby theory, it is believed that a liquid carrying an AC voltage iscapable of inducing the movement of electrons into and through alight-emitting device suspended in the liquid and not contacting the two115V AC power source leads due to the oscillating nature of electrons inan AC circuit, such that AC voltage in a liquid causes electrons to fillconductive paths connected to the liquid, in spite of the resistance ofthe conductive paths relative to the liquid, and will oscillate as an ACcurrent in those conductive paths, thereby performing work, e.g.,generating light in a light emitting diode. As a non-limiting example,light is produced in a liquid medium by a) placing a light-emittingdiode in a liquid comprising water and electrolytic ions, and b)inducing an AC voltage in the liquid, wherein c) the induction of ACvoltage in the electrolytic liquid causes the light-emitting diode togenerate light.

Through experimentation, Applicant unexpectedly discovered that one ormultiple light emitting units will emit light in a liquid when ACvoltage is applied to the liquid and when the light-emitting units arerated for voltages and amp draws commensurate with available electricalenergy. The production of autotrophic microorganisms is fundamentallyconstrained by the ability of light to penetrate through a liquid andthereby enable photosynthesis. Prior to Applicant's invention asdisclosed herein, no methods were believed to be known to produce lightin a liquid through the utilization of light-emitting devices physicallyunconnected to an electrical voltage source vis-à-vis solid conductivematerial. In one embodiment, the utilization of one or more, andpreferably many, free-floating light-emitting units in an electricallycharged liquid comprising autotrophic microorganisms enables a very highlight transmission efficiency, wherein previous light penetrationconstraints are largely overcome and high autotrophic microorganismdensities are fully enabled.

In several embodiments, a method for producing a polyhydroxyalkanoate(PHA) in a microorganism culture is provided. In some embodiments, themethod comprises the steps of: a) subjecting said culture to a growthperiod comprising exposing said culture to growth conditions to causesaid culture to reproduce, (b) subjecting said culture to apolymerization period comprising exposing said culture to polymerizationconditions to cause said culture to produce intracellular PHA, and (c)repeating step (a) and then second step (b) two or more times.

In some embodiments the growth period comprises a period in which theculture reproduces or otherwise produces biomass and/or reproduces. Insome embodiments, the polymerization period comprises a period in whichthe culture synthesizes PHA. In some embodiments, the growth period andthe polymerization period are induced by the culture media (e.g., theextracellular media around the culture). In some embodiments,alterations in the media conditions induce a transition (partial orcomplete) between growth and polymerization periods. In someembodiments, a culture is cycled between growth and polymerizationperiods two, three, four, or more times, in order to produce PHA andthen reproduce biomass, which is subsequently used to generateadditional PHA.

In some embodiments, the culture is exposed to non-sterile conditions.In certain such embodiments, input carbon is non-sterile. However, insome embodiments, sterile conditions exist. In some embodiments, theculture is dynamic over time in that it may be exposed to extraneousmicroorganisms. In certain embodiments, this is due to a non-sterileculture environment. In some embodiments, extraneous microorganismspotentiate the production of PHA.

In several embodiments, a process for the reduction of pigmentation in amicroorganism culture is provided. In one embodiment, the processcomprises the steps of providing a medium comprising a microorganismculture comprising dissolved oxygen, and increasing the concentration ofdissolved oxygen over successive periods to select for light-colored orlow-level pigmentation microorganisms.

PHA, while able to be treated post-production to reduce pigmentation, isless expensive to produce when lower levels of pigmentation exist. Somemicroorganisms are more pigmented than others, and therefore in severalembodiments, selection against the more pigmented microorganisms resultsin a less pigmented PHA, which reduces production costs. In someembodiments, the microorganisms cultured and selected for aremethanotrophic microorganisms. By manipulating the culture conditions,which benefit certain varieties of microorganisms, a less pigmentedculture (and hence a less pigmented PHA) result. In some embodiments,increases in concentration of dissolved oxygen over periods ranging from1-3 hours, 3-5 hours, 5-7 hours, 7-10 hours, 10-15 hours, or 15-24 hoursare used to select for less pigmented microorganisms. As discussedherein, in several embodiments, various compounding agents areoptionally added to the produced PHA (either during the production phaseor after production phase). Many such compounding agents, however,adversely affect the quality and/or performance (or othercharacteristic) of the final PHA product. In several embodiments,certain compounding agents, or combinations thereof, are added in orderto improve one or more characteristics of the final PHA product. In someembodiments, such agents aid in the reduction of pigmentation of thePHA. In some embodiments, compounding agents reduce the degree offiltration, centrifugation, settling (or other techniques disclosedherein to separate solids from liquid phases). Advantageously andunexpectedly, the addition of certain compounding agents, viewed in theart as harmful to the quality or to a certain characteristic of the PHA,the result achieve with the methods disclosed herein is a higher qualityPHA that is not adversely affected by the compounding agents, but ratheris enhanced by the addition of such compounds. [0113] In severalembodiments, a process for the conversion of a gas into apolyhydroxyalkanoate (PHA) is provided, wherein the process comprisesthe steps of: a) providing i) a first gas and ii) a culture ofmicroorganisms, b) contacting the first gas with the culture to causethe culture to convert the first gas into a second gas c) contacting thesecond gas with the culture, and d) causing the culture to use thesecond gas to produce PHA.

In some embodiments, the microorganisms are autotrophic, methanogenic,heterotrophic, or combinations thereof.

In one embodiment the first gas is carbon dioxide. In one embodiment thefirst gas is oxygen. In one embodiment the first gas is methane. In oneembodiment the first gas is a volatile organic compound.

In one embodiment the second gas is carbon dioxide. In one embodimentthe second gas is oxygen. In one embodiment the second gas is methane.In one embodiment the second gas is a volatile organic compound.

It shall be appreciated that the selection of the first and the secondgas is based on the type of microorganism or microorganisms beingcultured.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a block flow diagram comprising the steps of: microorganismfermentation and PHA synthesis, PHA-containing biomass removal,PHA-reduced biomass and isolated PHA production, PHA-reduced biomassrecycling and fermentation, and isolated PHA purification.

DETAILED DESCRIPTION

While PHAs have significant environmental advantages compared to fossilfuel-based plastics, the cost of PHA production is generally viewed as asignificant limitation to the industrial production and commercialadoption of PHAs. Generally, the overall cost of PHA production isdetermined by three major inputs: 1) carbon, 2) chemicals, and 3)energy. Accordingly, efforts to reduce the cost of PHA production mustaddress one or more of these areas, specifically by: i) reducing carboninput costs, ii) increasing carbon-to-PHA yields, iii) reducing thevolume of chemicals required for PHA production, and/or iv) increasingenergy-to-PHA yields.

As discussed above, food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems are widely regarded asthe most carbon, chemical, energy, and, thus, cost efficient PHAproduction method. Despite these efficiencies, sugar-based PHAproduction remains many times more expensive than fossil fuel-basedplastics production. Attempts to reduce the carbon input cost of the PHAproduction process, by utilizing carbon-containing industrial off-gases,such as carbon dioxide and methane, have been previously limited bytechnical challenges and stoichiometric limitations that render thegas-to-PHA production process significantly more energy and chemicalintensive, and thus more costly, than the food crop-based PHA productionprocess.

Specifically, these technical challenges and stoichiometric limitationsinclude: low mass transfer rates, low microorganism growth rates,extended polymerization times, low cell densities, high oxygen demand(relative to solid substrates), and low PHA cellular inclusionconcentrations. Whereas sugar-based fermentation systems have theability to generate high cellular densities and PHA inclusionconcentrations, carbon-containing gas-based fermentation processestypically cannot, based on fundamental cell morphology and mass transferconstraints, generate cellular and PHA densities exceeding 10-30% ofdensities possible in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon injection, oxygeninjection, system cooling, and culture mixing, as well as downstream PHApurification, significantly exceeds the energy-to-PHA ratio required forsugar-based PHA production methods, thereby rendering theemissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based PHAs.

Several embodiments of the present invention therefore relate to a novelmethod for the production of PHA using carbon-containing gases as asource of carbon (alone or in combination with a non-gaseous source ofcarbon), wherein the energy input-to-PHA production ratio, carboninput-to-PHA production ratio, and cost efficiency of the process issignificantly improved over previous gas-based PHA production processes.

In several embodiments, this process may be accomplished by a) culturinga first microorganism culture capable of metabolizing the carbon withinboth a carbon-containing gas and biomass, or a derivative thereof, b)manipulating the conditions of the culture to cause the culture toproduce PHA-containing biomass, c) removing a portion of thePHA-containing biomass; d) extracting at least a portion of the PHAwithin the removed PHA-containing biomass to create substantiallyisolated PHA and substantially PHA-reduced biomass, e) purifying theisolated PHA, and f) returning the PHA-reduced biomass to themicroorganism culture to cause the microorganism culture to metabolizethe carbon within the PHA-reduced biomass into PHA.

According to some embodiments, the steps of this process are as follows:(a) providing a microorganism culture comprising biomass and PHA; (b)removing a portion of the PHA-containing biomass from the culture, andextracting PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass; (c) purifying the isolated PHA,and (d) returning the PHA-reduced biomass to be mixed with the cultureto cause the culture to convert the carbon within the PHA-reducedbiomass into PHA. Each of the above recited steps in the process arediscussed in more detail below.

Providing a Microorganism Culture Comprising Biomass and PHA

The terms “microorganism”, “microorganisms”, “culture”, “cultures”, and“microorganism cultures”, as used herein, shall be given their ordinarymeanings and shall include, but not be limited to, a single strain ofmicroorganism and/or consortium of microorganisms, including, amongothers, genetically-engineered bacteria, fungi, algae, and/or yeast. Insome embodiments, microorganisms are naturally occurring and in someembodiments microorganisms are genetically-engineered. In someembodiments, both naturally occurring and genetically-engineeredmicroorganisms are used. In some embodiments, a mixed culture ofmicroorganisms may be used. In some embodiments, microorganisms orcultures shall include a microorganism metabolism system, including theinteractions and/or multiple functions of multiple cultures in one ormore conditions.

The terms “biomass” and “biomass material” shall be given their ordinarymeaning and shall include, but not be limited to, microorganism-derivedmaterial, including intracellular, cellular, and/or extracellularmaterial, such materials including, but not limited to, a polymer orpolymers, amino acids, nucleic acids, carbohydrates, lipids, sugars,PHA, volatile fatty acids, chemicals, gases, such as carbon dioxide,methane, volatile organic acids, and oxygen, and/or metabolicderivatives, intermediaries, and/or end-products. In severalembodiments, biomass is dried or substantially dried.

In some embodiments, the biomass contains less than about 99% water. Inother embodiments, the biomass contains between about 99% to about 75%water, including about 95%, 90%, 85%, and 80%. In some embodiments, thebiomass contains between about 75% and about 25% water, including75%-65%, 65%-55%, 55%-45%, 45%-35%, 35%-25%, and overlapping rangesthereof. In additional embodiments, the biomass contains from about 25%water to less than about 0.1% water, including 25%-20%, 20%-15%,15%-10%, 10%-5%, 5%-1%, 1%-0.1%, and overlapping ranges thereof. Instill other embodiments, the biomass contains no detectable amount ofwater. Depending on the embodiment, water is removed from the biomass byone or more of freeze drying, spray drying, fluid bed drying, ribbondrying, flocculation, pressing, filtration, and/or centrifugation. Insome embodiments, the biomass may be mixed with one or more chemicals(including, but not limited to solvents), such as methylene chloride,acetone, methanol, and/or ethanol, at various concentrations. In otherembodiments, the biomass may be processed through homogenization, heattreatment, pH treatment, enzyme treatment, solvent treatment, spraydrying, freeze drying, sonication, or microwave treatment. As usedherein, the term “PHA-reduced biomass” shall be given its ordinarymeaning and shall mean any biomass wherein at least a portion of PHA hasbeen removed from the biomass through a PHA extraction process. As usedherein, the term “PHA-containing biomass” shall be given its ordinarymeaning and shall mean any biomass wherein at least a portion of thebiomass is PHA.

Microorganism cultures useful for the invention described herein includea single strain, and/or a consortium of strains, which are individuallyand/or collectively capable of using carbon containing gases andbiomass, including PHA-reduced biomass, as a source of carbon for theproduction of biomass and PHA. In some embodiments, a microorganismculture according to several embodiments, comprises a microorganismculture that utilizes PHA-reduced biomass, or any derivative thereof,including methanotrophic microorganisms, anaerobic digestion cultures,and other heterotrophic microorganisms, as a source of carbon for theproduction of biomass, or metabolic derivatives including, and inparticular, the production of PHA, protein, methane, and/or carbondioxide (herein, “biomass-utilizing microorganisms”). As used herein,the terms “microorganism”, “culture”, “microorganism culture,”“microorganism system,” “microorganism consortium,” and “consortium ofmicroorganisms” are used interchangeably. Additionally, any of theseterms may refer to one, two, three, or more microorganism culturesand/or strains, including a microorganism system that is collectivelycapable of carrying out a complex metabolic function (e.g., conversionof PHA-reduced biomass to methane, carbon dioxide, protein, and/or PHA).In several embodiments, the microorganism culture comprises of aconsortium of carbon-containing gas-utilizing microorganisms and aconsortium of biomass-utilizing microorganisms. In some embodiments, thegases metabolized by such cultures comprise methane, carbon dioxide,and/or a combination thereof.

In some embodiments, the microorganism culture comprises a consortium ofacidogenic, acetogenic, methanogenic, methanotrophic, and/or autotrophicmicroorganisms in one or more individual bioreactors. As such, in someembodiments, the cultures are grown in one or more distinct cultureconditions. In some embodiments, the conditions are either aerobic oranaerobic conditions. In some embodiments, culture conditions are variedover time (e.g. initially aerobic with a transition to anaerobic, orvice versa). As used herein, the term “bioreactor” shall be given itsordinary meaning and shall also refer to a tank, vessel, or anycontainer or device suitable for growth and culturing of microorganisms.

In some embodiments, the microorganism culture is contained within asingle vessel, wherein the steps of converting PHA-reduced biomass tobiomass, converting biomass to PHA, and converting carbon-containinggases to biomass and/or PHA occur simultaneously or sequentially.

In other embodiments, the microorganism culture is contained withinmultiple vessels, which are designed to carry out specific and uniquefunctions. For example, one embodiment includes the steps of (a)converting PHA-reduced biomass to PHA-reduced biomass-derived materialssuch volatile organic acids, methane, and/or carbon dioxide, which iscarried out in a first vessel and (b) synthesizing PHA from PHA-reducedbiomass-derived materials which is carried out in a second, separatetank under independent conditions. In some embodiments, one or more ofthe tanks is an anaerobic digestion tank and one or more other tank isan aerobic fermentation tank.

As used herein, the term “gas-utilizing microorganisms” shall be givenits ordinary meaning and shall refer to microorganisms capable ofutilizing gases containing carbon for the production of biomass,including the production of PHA. Similarly, the terms “methanotrophicmicroorganisms” and “methane-utilizing microorganisms” shall be giventheir ordinary meanings and shall refer to microorganisms capable ofutilizing methane as a source of carbon for the production of biomass.Further, the terms “autotrophic microorganisms” and “carbondioxide-utilizing microorganisms” shall be given their ordinary meaningand shall refer to microorganisms capable of utilizing carbon dioxide asa source of carbon for the production of biomass, includingmicroorganisms that utilize natural and/or synthetic sources of light tocarry out the metabolism of carbon dioxide into biomass. The term“heterotrophic microorganisms”, as used herein, shall be given itsordinary meaning and shall include methanotrophic, methanogenic,acidogenic, acetogenic and biomass-utilizing microorganisms, includingmicroorganisms that convert sugar, volatile fatty acids, or other carbonsubstrates to biomass. The term “methanogenic microorganisms” shall begiven its ordinary meaning and shall refer to microorganisms thatconvert biomass to methane, including the consortium of microorganismsrequired to carry out such a process, including, but not limited to,acidogenic and acetogenic microorganisms.

As discussed herein, in several embodiments, carbon-containing gases areused as a source of carbon by microorganism cultures. In someembodiments, other sources of carbon are used (e.g., PHA-reducedbiomass), either alone or in combination with carbon-containing gases.In some embodiments, the carbon-containing gases used include, but arenot limited to, carbon dioxide, methane, ethane, butane, propane,benzene, xylene, acetone, methylene chloride, chloroform, volatileorganic compounds, hydrocarbons, and/or combinations thereof. The sourceof the carbon-containing gases depends on the embodiment. For example,carbon-containing gas sources used in some embodiments includelandfills, wastewater treatment plants, anaerobic metabolism, powerproduction facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, cement production facilities, and/oranaerobic organic material digesters, including both solid and liquidmaterial digesters.

In several embodiments described herein, microorganisms may include, butare not limited to, yeast, fungi, algae, and bacteria (includingcombinations thereof). Suitable yeasts include, but are not limited to,species from the genera Candida, Hansenula, Torulopsis, Saccharomyces,Pichia, 1-Debaryomyces, Lipomyces, Cryptococcus, Nematospora, andBrettanomyces. Suitable genera include Candida, Hansenula, Torulopsis,Pichia, and Saccharomyces. Non-limiting examples of suitable speciesinclude, but are not limited to: Candida boidinii, Candida mycoderma,Candida utilis, Candida stellatoidea, Candida robusta, Candidaclaussenii, Candida rugosa, Brettanomyces petrophilium, Hansenulaminuta, Hansenula satumus, Hansenula californica, Hansenula mrakii,Hansenula silvicola, Hansenula polymorpha, Hansenula wickerhamii,Hansenula capsulata, Hansenula glucozyma, Hansenula henricii, Hansenulanonfermentans, Hansenula philodendra, Torulopsis candida, Torulopsisbolmii, Torulopsis versatilis, Torulopsis glabrata, Torulopsismolishiana, Torulopsis nemodendra, Torulopsis nitratophila, Torulopsispinus, Pichia farinosa, Pichia polymorpha, Pichia membranaefaciens,Pichia pinus, Pichia pastoris, Pichia trehalophila, Saccharomycescerevisiae, Saccharomyces fragilis, Saccharomyces rosei, Saccharomycesacidifaciens, Saccharomyces elegans, Saccharomyces rouxii, Saccharomyceslactis, and/or Saccharomyces fractum.

Suitable bacteria include, but are not limited to, species from thegenera Bacillus, Mycobacterium, Actinomyces, Nocardia, Pseudomonas,Methanomonas, Protaminobacter, Methylococcus, Arthrobacter,Methylomonas, Brevibacterium, Acetobacter, Methylomonas, Brevibacterium,Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium,Rhodopseudomonas, Microbacterium, Achromobacter, Methylobacter,Methylosinus, and Methylocystis. Preferred genera include Bacillus,Pseudomonas, Protaminobacter, Micrococcus, Arthrobacter and/orCorynebacterium. Non-limiting examples of suitable species include, butare not limited to: Bacillus subtilus, Bacillus cereus, Bacillus aureus,Bacillus acidi, Bacillus urici, Bacillus coagulans, Bacillus mycoides,Bacillus circulans, Bacillus megaterium, Bacillus licheniformis,Pseudomonas ligustri, Pseudomonas orvilla, Pseudomonas methanica,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas oleovorans,Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas pyocyanea,Pseudomonas methylphilus, Pseudomonas brevis, Pseudomonas acidovorans,Pseudomonas methanoloxidans, Pseudomonas aerogenes, Protaminobacterruber, Corynebacterium simplex, Corynebacterium hydrocarbooxydans,Corynebacterium alkanum, Corynebacterium oleophilus, Corynebacteriumhydrocarboclastus, Corynebacterium glutamicum, Corynebacterium viscosus,Corynebacterium dioxydans, Corynebacterium alkanum, Micrococcuscerificans, Micrococcus rhodius, Arthrobacter rufescens, Arthrobacterparafficum, Arthrobacter citreus, Methanomonas methanica, Methanomonasmethanooxidans, Methylomonas agile, Methylomonas albus, Methylomonasrubrum, Methylomonas methanolica, Mycobacterium rhodochrous,Mycobacterium phlei, Mycobacterium brevicale, Nocardia salmonicolor,Nocardia minimus, Nocardia corallina, Nocardia butanica,Rhodopseudomonas capsulatus, Microbacterium ammoniaphilum,Archromobacter coagulans, Brevibacterium butanicum, Brevibacteriumroseum, Brevibacterium flavum, Brevibacterium lactofermentum,Brevibacterium paraffinolyticum, Brevibacterium ketoglutamicum, and/orBrevibacterium insectiphilium.

In several embodiments, more than one type or species of microorganismis used. For example, in some embodiments, both algae and bacteria areused. In some embodiments, several species of yeast, algae, fungi,and/or bacteria are used. In some embodiments, a single yeast, algae,fungi, and/or bacteria species is used. In some embodiments, aconsortium of cyanobacteria is used. In some embodiments, a consortiumof methanotrophic microorganisms is used. In still additionalembodiments, a consortium of both methanotrophic bacteria andcyanobacteria are used. In several embodiments, methanotrophic,heterotrophic, methanogenic, and/or autotrophic microorganisms are used.

In several embodiments of the invention, the microorganism culturecomprises a consortium of methanotrophic, autotrophic, and/orheterotrophic microorganisms, wherein methane and/or carbon dioxide isindividually, interchangeably, or simultaneously utilized for theproduction of biomass. In some embodiments, PHA-reduced biomass is usedas a source of carbon by heterotrophic, autotrophic, and/ormethanotrophic microorganisms. In several embodiments of the invention,the microorganism culture comprises methanotrophic microorganisms,cyanobacteria, and non-methanotrophic heterotrophic microorganisms,wherein methane and carbon dioxide are continuously utilized as sourcesof carbon for the production of biomass and PHA.

In some embodiments, microorganisms are employed in a non-sterile, open,and/or mixed environment. In other embodiments, microorganisms areemployed in a sterile and/or controlled environment.

The terms “PHA”, “PHAs”, and “polyhydroxyalkanoate”, as used herein,shall be given their ordinary meaning and shall include, but not belimited to, polymers generated by microorganisms as energy and/or carbonstorage vehicles; biodegradable and biocompatible polymers that can beused as alternatives to petrochemical-based plastics such aspolypropylene, polyethylene, and polystyrene; polymers produced bybacterial fermentation of sugars, lipids, or gases; and/or thermoplasticor elastomeric materials derived from microorganisms. PHAs include, butare not limited to, polyhydroxybutyrate (PHB), polyhydroxyvalerate(PHV), polyhydroxybutyrate-covalerate (PHBV), and polyhydroxyhexanoate(PHHx), as well as both short chain length (SCL), medium chain length(MCL), and long chain length (LCL) PHAs.

The terms “growth-culture medium”, “growth medium”, “growth-culturemedia”, “medium”, and “media”, as used herein shall be given theirordinary meaning and shall also refer to materials affecting the growth,metabolism, PHA synthesis, and/or reproductive activities ofmicroorganisms. Non-limiting examples of growth-culture media used inseveral embodiments include a mineral salts medium, which may comprisewater, nitrogen, vitamins, iron, phosphorus, magnesium, and variousother nutrients suitable to effect, support, alter, modify, control,constrain, and/or otherwise influence the metabolism and metabolicorientation of microorganisms. A growth-culture medium may comprisewater filled with a range of mineral salts. For example, each liter of aliquid growth-culture medium may be comprised of about 0.7-1.5 g KH₂PO₄,0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃, 0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28mg CaCl₂*2H₂O, 5.0-5.4 mg EDTA Na₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O,0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mg MnCl₂*2H₂O, 0.06-0.08 mg ZnCl₂,0.05-0.07 mg H₃BO₃, 0.023-0.027 mg NiCl₂*6H₂O, 0.023-0.027 mgNaMoO₄*2H₂O, and 0.011-0.019 mg CuCl₂*2H₂O. A growth-culture medium canbe of any form, including a liquid, semi-liquid, gelatinous, gaseous,foam, or solid substrate.

In several embodiments of the invention, a microorganism culture isproduced in a liquid growth medium, wherein carbon dioxide and methaneare utilized as a gaseous source of carbon for the production ofmethanotrophic and/or autotrophic biomass. In some embodiments,PHA-reduced methanotrophic and/or PHA-reduced autotrophic biomass isutilized as a source of carbon for the production of heterotrophicbiomass and heterotrophically-produced PHA. In some embodiments, thegrowth medium is manipulated to effect the growth, reproduction, and PHAsynthesis of the microorganism culture. Methods for the production ofmethanotrophic microorganisms are disclosed in the art, and aredescribed by Herrema, et al., in U.S. Pat. No. 7,579,176, which ishereby incorporated by reference in its entirety. Methods for theproduction of cyanobacteria are described by Lee, et al. (“High-densityalgal photobioreactors using light-emitting diodes,” Biotechnology andBioengineering, Vol. 44, Issue 10, pp. 1161-1167), which is herebyincorporated by reference in its entirety. Methods for the production ofmethane from biomass are described by Deublein, et al. (“Biogas fromWaste and Renewable Resources, WILEY-VCH Verlag GmbH & Co. KgaA, 2008),which is hereby incorporated by reference in its entirety. In someembodiments, PHA synthesis may be effected through the manipulation ofone of more elements of the culture medium, including through thereduction, increase, or relative change in either the total orbioavailable concentration of one or more of the following elements(also referred to as essential nutrients): carbon, oxygen, magnesium,phosphorus, phosphate, potassium, sulfate, sulfur, calcium, boron,aluminum, chromium, cobalt, iron, copper, nickel, manganese, molybdenum,sodium, nitrogen, nitrate, ammonia, ammonium, urea, amino acids,methane, carbon dioxide, and/or hydrogen. Methods for the production ofPHA are described by Herrema, et al., in U.S. Pat. No. 7,579,176. Itshall be appreciated that all of the various components (includingelements, compounds, liquids, gases, solids, and other compositions) ofa culture medium can be considered essential nutrients, given that theysupport the growth of the microorganisms.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is effected by manipulating the concentration one or more elementswithin a growth medium selected from the group consisting of: nitrogen,methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium, iron,copper, sulfate, manganese, calcium, chlorine, boron, zinc, aluminum,nickel, and/or sodium, and combinations thereof. Methods to control theconcentration of elements within the medium include, but are not limitedto, automatic, continuous, batch, semi-batch, manual, injection, solidfeed, liquid, or other methods of inputting one or more chemical intothe medium, wherein the total and/or bioavailable concentration ofelements is increased, decreased, maintained, adjusted, or otherwisecontrolled at one or more time and/or physical chemical adjustmentpoints. Additional methods to adjust the total or bioavailableconcentration of one or more elements within a mineral media include,but are not limited to, the directed precipitation, chelation,de-chelation, and de-precipitation of elements. In one embodiment, thedirected precipitation or chelation of one or more element is utilizedto reduce the total or bioavailable concentration of one or more elementwithin a medium and thereby induce or increase PHA production in abiomass. In one embodiment, an ion exchange system, including one ormore reversible ion exchange resins, is used to control theconcentration of ions with the medium and/or control the precipitationor solubilization of elements within the medium. In one embodiment, themedium is passed through an ion exchange resin in order to induce thereduction or increase of a specific ion in the medium in order to induceor preclude PHA production in a biomass.

In one embodiment, the concentration of one or more elements within agrowth culture medium is increased, controlled, manipulated, or managedto induce or increase the rate of PHA production in biomass, including amicroorganism culture. As used herein, the terms “control”,“manipulate”, “adjust”, “maintain”, “manage” and the like shall be giventheir ordinary meaning and shall also refer to steps which are taken tokeep or achieve concentrations of certain nutrients, compounds orelements of a culture within a desired range. For example, in onecontext controlling the concentration of an element may result in themaintenance of the concentration of that element within a certain range,that concentration being achieved by the addition of that element and/ordilution of the culture to reduce the concentration of that element. Inone embodiment, the concentration of phosphorus within the medium isincreased to induce or increase the rate of PHA production in amicroorganism culture. In another embodiment, the concentration of anelement, e.g., phosphorus, carbon dioxide, iron, copper, oxygen,methane, and/or magnesium, within the medium is manipulated or increasedto cause a metabolic shift in the microorganism culture, such that theproduction of non-PHA materials by the culture using nitrogen sources(e.g., nitrate, ammonia, ammonium, dinitrogen, urea, or amino acids) isreduced, inhibited, or otherwise impacted to enhance PHA production. Inone embodiment, the concentration of phosphorus within the medium ismanipulated or increased to reduce the utilization of nutrients,including nitrogen, oxygen, and/or carbon, for the production of non-PHAmaterials by the culture. In another embodiment, the concentration ofphosphorus within the medium is manipulated or increased to reduce theutilization of nutrients for the production of non-PHA materials by theculture and induce or increase the rate of PHA production in theculture. In some embodiments, an increase in the concentration ofphosphorus causes a metabolic shift that favors the production of PHA atthe expense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars, lipids,particularly but not necessarily under growth-limiting conditions,including nitrogen (e.g., nitrate, ammonia, ammonium, dinitrogen, urea,or amino acids), oxygen, magnesium, potassium, iron, copper, or othernutrient-limiting conditions. In some embodiments, an increase in theconcentration of phosphorus above 0.00 ppm, 0.01 ppm, 0.02 ppm, 0.05ppm, 0.10 ppm, 0.20 ppm, 0.50 ppm, 1.00 ppm, 1.25 ppm, 1.50 ppm, 1.75ppm, 2.00 ppm, 2.20 ppm, 2.40 ppm, 3 ppm, 4 ppm 5 ppm, 6 ppm, 8 ppm, 20mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, or120 mM, and particularly above 40 mM or 80 mM, causes a reduction in theutilization of carbon and nitrogen sources for the production of non-PHAmaterial, including proteins, non-PHA polymers, nucleic acids, lipids,pigments, polysaccharides, methanobactin, and/or carbon dioxide, and,under growth-limiting conditions, an increase in the utilization ofcarbon sources for the production of PHA material, as a result ofmetabolic changes in the culture and/or chemical interactions betweenchemicals within the media and/or culture induced by augmentedconcentrations of phosphorus. The elevation of phosphorus concentrationsas a method to induce or increase the rate of PHA production in abiomass culture is contrary to the teachings of the prior art, whichteaches that PHA production is induced or enhanced by reducing oreliminating the concentration of elements, such as, e.g., phosphorus inthe mineral medium. The addition or controlled elevation of an element,such as, e.g., phosphorus to a biomass system to induce or increase PHAproduction produces an unexpected and surprising increase in PHAproduction in a biomass system. An element such as, e.g., phosphorus maybe added to the mineral media using a variety of phosphorus sources,including phosphorus, phosphate, phosphoric acid, sodium phosphate,disodium phosphate, monosodium phosphate, and/or potassium phosphate;other elements, such as dissolved carbon dioxide, Fe(II) iron, Fe(III)iron, copper sulfate, Fe-EDTA, dissolved oxygen, dissolved methane,and/or magnesium sulfate, hydrogen sulfide, and sodium hydroxide areamong other potential sources of elements that may be added to themineral media.

In one embodiment of the invention, the concentrations of dissolvedgases, such as methane, oxygen, carbon dioxide, and/or nitrogen, aremanipulated to increase the rate of PHA production relative to the rateof cellular production of non-PHA materials and, specifically, to causea reduction in the utilization of carbon or nitrogen sources for theproduction of non-PHA material, including proteins, non-PHA polymers,enzymes, nucleic acids, lipids, pigments, polysaccharides, and/or carbondioxide, and, under some conditions, including growth-limitingconditions, further cause an increase in the utilization of carbonsources for the production of PHA material, as a result of metabolicchanges in the culture and/or chemical interactions between chemicalswithin the media and/or culture induced by augmented concentrations ofone or more of such dissolved gases. In one embodiment, theconcentration of methane or dissolved methane is manipulated to betweenabout 0.01 ppm and about 0.05 ppm, between about 0.05 ppm and about 0.1ppm, between about 0.1 ppm and about 0.5 ppm, between about 0.5 ppm andabout 1.0 ppm, between about 1.0 ppm and about 1.5 ppm, between about1.5 ppm and about 1.75 ppm, between about 1.75 ppm and about 2.0 ppm,between about 2.0 ppm and about 2.5 ppm, between about 2.5 ppm and about3.0 ppm, between about 3.0 ppm and about 3.5 ppm, between about 3.5 ppmand about 4.0 ppm, between about 4.0 ppm and about 4.5 ppm, betweenabout 4.5 ppm and about 5.0 ppm, between about 5.0 ppm and about 6.0ppm, between about 6.0 ppm and about 7.0 ppm, between about 7.0 ppm andabout 8.0 ppm, between about 8.0 ppm and about 10 ppm, between about 10ppm and about 15 ppm, between about 15 ppm and about 20 ppm, betweenabout 20 ppm and about 30 ppm, or between about 30 ppm and about 50 ppm(and overlapping ranges thereof) to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inone embodiment, the concentration of oxygen or dissolved oxygen ismanipulated to between about 0.01 ppm and about 0.05 ppm, between about0.05 ppm and about 0.1 ppm, between about 0.1 ppm and about 0.5 ppm,between about 0.5 ppm and about 1.0 ppm, between about 1.0 ppm and about1.5 ppm, between about 1.5 ppm and about 1.75 ppm, between about 1.75ppm and about 2.0 ppm, between about 2.0 ppm and about 2.5 ppm, betweenabout 2.5 ppm and about 3.0 ppm, between about 3.0 ppm and about 3.5ppm, between about 3.5 ppm and about 4.0 ppm, between about 4.0 ppm andabout 4.5 ppm, between about 4.5 ppm and about 5.0 ppm, between about5.0 ppm and about 6.0 ppm, between about 6.0 ppm and about 7.0 ppm,between about 7.0 ppm and about 8.0 ppm, between about 8.0 ppm and about10 ppm, between about 10 ppm and about 15 ppm, between about 15 ppm andabout 20 ppm, between about 20 ppm and about 30 ppm, or between about 30ppm and about 50 ppm (and overlapping ranges thereof) to reduce theproduction of non-PHA materials relative to the production of PHAmaterials in a culture. In another embodiment, the concentration ofcarbon dioxide or dissolved carbon dioxide is manipulated to betweenabout 0.01 ppm and about 0.05 ppm, between about 0.05 ppm and about 0.1ppm, between about 0.1 ppm and about 0.5 ppm, between about 0.5 ppm andabout 1.0 ppm, between about 1.0 ppm and about 1.5 ppm, between about1.5 ppm and about 1.75 ppm, between about 1.75 ppm and about 2.0 ppm,between about 2.0 ppm and about 2.5 ppm, between about 2.5 ppm and about3.0 ppm, between about 3.0 ppm and about 3.5 ppm, between about 3.5 ppmand about 4.0 ppm, between about 4.0 ppm and about 4.5 ppm, betweenabout 4.5 ppm and about 5.0 ppm, between about 5.0 ppm and about 6.0ppm, between about 6.0 ppm and about 7.0 ppm, between about 7.0 ppm andabout 8.0 ppm, between about 8.0 ppm and about 10 ppm, between about 10ppm and about 15 ppm, between about 15 ppm and about 20 ppm, betweenabout 20 ppm and about 30 ppm, or between about 30 ppm and about 50 ppmbetween about 50 ppm and about 100 ppm, between about 100 ppm and about200 ppm, between about 200 ppm and about 500 ppm, between about 500 ppmand about 1000 ppm, between about 100 ppm and about 1500 ppm, betweenabout 1500 ppm and about 2000 ppm, between about 2000 ppm and about 3000ppm, between about 3000 ppm and about 5000 ppm, between about 5000 ppmand about 10,000 ppm, between about 10,000 ppm and about 20,000 ppm (andoverlapping ranges thereof) to reduce the production of non-PHAmaterials relative to the production of PHA materials in a culture. Inanother embodiment, the concentration of nitrogen or dissolved nitrogenis manipulated to above at least between about 0.01 ppm and about 0.05ppm, between about 0.05 ppm and about 0.1 ppm, between about 0.1 ppm andabout 0.5 ppm, between about 0.5 ppm and about 1.0 ppm, between about1.0 ppm and about 1.5 ppm, between about 1.5 ppm and about 1.75 ppm,between about 1.75 ppm and about 2.0 ppm, between about 2.0 ppm andabout 2.5 ppm, between about 2.5 ppm and about 3.0 ppm, between about3.0 ppm and about 3.5 ppm, between about 3.5 ppm and about 4.0 ppm,between about 4.0 ppm and about 4.5 ppm, between about 4.5 ppm and about5.0 ppm, between about 5.0 ppm and about 6.0 ppm, between about 6.0 ppmand about 7.0 ppm, between about 7.0 ppm and about 8.0 ppm, betweenabout 8.0 ppm and about 10 ppm, between about 10 ppm and about 15 ppm,between about 15 ppm and about 20 ppm, between about 20 ppm and about 30ppm, or between about 30 ppm and about 50 ppm, and overlapping rangesthereof, to reduce the production of non-PHA materials relative to theproduction of PHA materials in a culture. Without being limited bytheory, it is believed that, in some metabolic pathways, an increase inthe concentration of methane, oxygen, carbon dioxide, and/or nitrogencauses a metabolic shift that favors the production of PHA at theexpense of other non-PHA materials, including a reduction in theproduction of protein, nucleic acids, polysaccharides, sugars, and/orlipids, particularly, but not necessarily, under growth-limiting, thatis, PHA synthesis, conditions. In one embodiment, the synthesis ofpolyhydroxyalkanote (PHA) in a biomass material is effected, comprisingthe steps of: (a) providing a medium comprising a biomass metabolizing asource of carbon, and (b) increasing or maintaining above a minimum theconcentration of an element in the medium to cause the biomass tosynthesize PHA or increase the synthesis rate of PHA relative to thesynthesis rate of non-PHA material. In one embodiment, the PHA ispolyhydroxybutyrate (PHB). In one embodiment, the biomass is one or moremicroorganisms. In one embodiment, the step of increasing theconcentration of the element in the medium causes a reduction in theconcentration of sugar, lipids, nucleic acids, saccharides,polysaccharides, and/or pigments in the biomass relative to theconcentration of PHA in the biomass. In one embodiment, the element isone or more of the following: phosphorus, phosphate, phosphoric acid,sodium phosphate, disodium phosphate, monosodium phosphate, or potassiumphosphate, methane, oxygen, carbon dioxide, hydroxyl ions, hydrogen ion,nitrogen. In one embodiment, the element is one or more of thefollowing: EDTA, citric acid, iron, copper, magnesium, manganese, zinc,calcium, potassium, boron. In one embodiment, the PHA synthesis rate isincreased relative to the synthesis rate of PHA in said biomass in theabsence of said. In one embodiment, the synthesis of polyhydroxyalkanote(PHA) in a biomass material is effected, comprising the steps of: (a)providing a medium comprising a biomass and an element, and (b)maintaining above a minimum concentration or increasing theconcentration of the element in the medium to cause the biomass materialto metabolically synthesize PHA at the expense of alternative biomassenergy and/or carbon storage materials. In one embodiment, the elementis phosphorus, oxygen, magnesium, calcium, copper, iron, methane, carbondioxide, or nitrogen. In one embodiment, the element is phosphorus. Inone embodiment, the element is oxygen. In one embodiment, the element ismagnesium. In one embodiment, the element is calcium. In one embodiment,the element is copper. In one embodiment, the element is iron. In oneembodiment, the biomass comprises one or more microorganisms. In oneembodiment, one or more microorganisms comprise methanotrophicmicroorganisms. In one embodiment, one or more microorganisms compriseheterotrophic microorganisms. In one embodiment, one or moremicroorganisms comprise autotrophic microorganisms. In one embodiment,one or more microorganisms comprise methanogenic microorganisms.

In one embodiment, the invention comprises manipulating theconcentration of elements, e.g., copper, iron, phosphorus, oxygen,methane, carbon dioxide, in the culture medium to control theconcentration of sMMO and/or pMMO produced by a methanotrophic culturein order to control the relative ratio of sMMO to pMMO in the cultureand thereby control the growth conditions, metabolic status, metabolicdisposition, and/or specification of PHA produced by the culture. Insome embodiments, sMMO is expressed in a range between about 0% and 100%of a methanotrophic culture by dry cell weight, as a percentage ofmicroorganisms expressing sMMO, or as a percentage of total MMOexpressed by one or more methanotrophic cells, including between 0% and1%, between about 1% and about 2%, between about 2% and about 3%,between about 3% and about 5%, between about 5% and about 10%, betweenabout 10% and about 20%, between about 20% and about 30%, between about30% and about 50%, between about 50% and about 70%, between about 70%and about 80%, between about 80% and about 90%, between about 90% andabout 95%, between about 95% and about or 100%, and overlapping rangesthereof. Simultaneously, or independently, in some embodiments, pMMO isexpressed in a range between about 0% and 100% of a methanotrophicculture by dry cell weight, as a percentage of microorganisms expressingpMMO, or as a percentage of total MMO expressed by one or moremethanotrophic cells, including between 0% and 1%, between about 1% andabout 2%, between about 2% and about 3%, between about 3% and about 5%,between about 5% and about 10%, between about 10% and about 20%, betweenabout 20% and about 30%, between about 30% and about 50%, between about50% and about 70%, between about 70% and about 80%, between about 80%and about 90%, between about 90% and about 95%, between about 95% andabout or 100%, and overlapping ranges thereof. In some embodiments, theratio of sMMO to pMMO produced in a methanotrophic culture is controlledto control the specification of PHA produced by a culture In someembodiments, the relative weight ratio of sMMO to pMMO in amethanotrophic culture is at least or approximately 0 to 1,approximately 0.0000001 to 1, approximately 0.0001 to 1, approximately0.001 to 1, approximately 0.01 to 1, approximately 0.1 to 1,approximately 1 to 1, approximately 2 to 1, approximately 3 to 1,approximately 5 to 1, approximately 10 to 1, approximately 15 to 1,approximately 20 to 1, approximately 25 to 1, approximately 30 to 1,approximately 35 to 1, approximately 50 to 1, approximately 65 to 1,approximately 70 to 1, approximately 80 to 1, approximately 90 to 1,approximately 95 to 1, approximately 98 to 1, approximately 99 to 1,approximately 100 to 1, approximately 1000 to 1, approximately 10,000 to1, approximately 100,000 to 1, or approximately 1,000,000 to 1,respectively. In some embodiments, the relative weight ratio of pMMO tosMMO in a methanotrophic culture is approximately 0 to 1, approximately0.0000001 to 1, approximately 0.0001 to 1, approximately 0.001 to 1,approximately 0.01 to 1, approximately 0.1 to 1, approximately 1 to 1,approximately 2 to 1, approximately 3 to 1, approximately 5 to 1,approximately 10 to 1, approximately 15 to 1, approximately 20 to 1,approximately 25 to 1, approximately 30 to 1, approximately 35 to 1,approximately 50 to 1, approximately 65 to 1, approximately 70 to 1,approximately 80 to 1, approximately 90 to 1, approximately 95 to 1,approximately 98 to 1, approximately 99 to 1, approximately 100 to 1,approximately 1000 to 1, approximately 10,000 to 1, approximately100,000 to 1, or approximately 1,000,000 to 1. In some embodiments,controlling the relative concentrations of sMMO and pMMO produced by aculture of methanotrophic microorganisms, it is possible to control themetabolic status of the culture and thereby control the type of PHA andother cellular material produced by the culture, particularly in thepresence of volatile organic compounds, fatty acids, volatile fattyacids, methanol, formate, acetate, dissolved carbon dioxide, dissolvedmethane, dissolved oxygen, and other elements or compounds that impactthe metabolism of a culture of methanotrophic microorganisms in acertain manner according to the relative concentration of sMMO or pMMOin such a culture. In some embodiments, sMMO and/or pMMO is expressed ina range between about 0% and 100% of a methanotrophic culture by drycell weight, as a percentage of microorganisms expressing sMMO or pMMO,or as a percentage of total MMO expressed by one or more methanotrophiccells, including between 0% and 1%, between about 1% and about 2%,between about 2% and about 3%, between about 3% and about 5%, betweenabout 5% and about 10%, between about 10% and about 20%, between about20% and about 30%, between about 30% and about 50%, between about 50%and about 70%, between about 70% and about 80%, between about 80% andabout 90%, between about 90% and about 95%, between about 95% and aboutor 100%, and overlapping ranges thereof prior to, during, throughout, orafter a PHA production phase. In one embodiment, sMMO is not expressed,or is expressed in low concentrations, in a methanotrophic culture priorto, during, throughout, or after a PHA production phase. Without beinglimited by theory, it is believed that the directed or controlledabsence or reduction of sMMO in a methanotrophic culture producing PHA,particularly in the presence of non-methane organic compounds that canbe metabolized by methanotrophic microorganisms, engenders PHAproduction stability, consistency, and control by selectively shieldingagainst the metabolism of one or some or many non-methane compounds thatmight otherwise be metabolized in the presence of sMMO, which enablesthe metabolism of a larger group of non-methane compounds than pMMO.Similarly, in one embodiment, pMMO is not expressed, or is expressed inlow concentrations, in a methanotrophic culture prior to, during,throughout, or after a PHA production phase. In some embodiments, thedirected or controlled absence or reduction of pMMO in a methanotrophicculture producing PHA, particularly in the presence of non-methaneorganic compounds that can be metabolized by methanotrophicmicroorganisms, engenders PHA production stability, consistency, andcontrol by selectively inducing or promoting the metabolism of one orsome or many non-methane compounds that might otherwise be not bemetabolized using pMMO. In some embodiments, in some methanotrophiccultures, sMMO promotes PHA synthesis at high intracellularconcentrations by reducing cellular production of non-PHA materials,particularly as compared to PHA synthesis using pMMO. By controlling theconcentration of sMMO relative to pMMO in a methanotrophic microorganismculture in the presence of methane and/or non-methane organic compounds,including VOCs and other carbon-containing materials, such as volatilefatty acids, acetone, acetic acid, acetate, formate, formic acid,chloroform, methylene chloride, carbon dioxide, ethane, and/or propane,it is possible to control the specification or type of PHA produced bythe culture, including the molecular weight, polydispersity, melt flow,impact strength, and other similar functional characteristics. In someembodiments, it is preferable to maintain the concentration of copper inthe culture media in order to promote sMMO production. In someembodiments, in some methanotrophic cultures, the production of sMMO inmany, most, or substantially all of the methanotrophic cells enables theculture to produce more PHA when subject to a nutrient limiting stepthan would otherwise be produced if the relative ratio of pMMO in theculture was higher prior to the nutrient limiting step. Conversely, insome embodiments, it is preferable to maintain the concentration ofcopper in the culture media in order to promote pMMO production. In someembodiments, in some methanotrophic cultures, the production of pMMO inmany, most, or substantially (e.g., more than 50%, more than 60%, morethan 70%, more than 80%, more than 90%, or more) all of themethanotrophic cells enables the culture to produce more PHA whensubject to a nutrient limiting step than would otherwise be produced ifthe relative ratio of sMMO in the culture was higher prior to thenutrient limiting step. In one embodiment, one or more methanotrophiccells or cultures are subject to repeated growth and PHA synthesiscycles or steps, wherein the relative concentration of sMMO to pMMO inthe cells or cultures are caused to remain approximately similar or thesame in each new cycle or step in order to control the specificationand/or functionality of the PHA produced by the culture. In severalembodiments, the relative concentration of sMMO to pMMO is variableacross the repetitions. In some embodiments, the ratio of relativeconcentrations decreases (e.g., there is progressively less sMMO andprogressively more pMMO with each repetition). In one embodiment, thefunctional characteristics of PHA produced by a methanotrophic cultureexposed to methane emissions comprising methane, carbon dioxide, and oneor more volatile organic compounds are controlled and optimized, whereinthe method comprises: (a) providing a methanotrophic culture in amineral medium comprising nutrients, (b) controlling the concentrationof one or more of nutrients in the medium to cause the culture toproduce a defined relative ratio of sMMO to pMMO, (c) and controllingthe concentration of the nutrients in the medium to cause the culture toproduce PHA. In one embodiment, the culture produces essentially onlypMMO. As used herein, the term “essentially only” shall be given itsordinary meaning and shall also refer to production of pMMO or sMMO inan amount greater than about 50%, greater than about 55%, greater thanabout 60%, greater than about 65%, greater than about 70%, greater thanabout 75%, greater than about 80%, greater than about 85%, greater thanabout 90%, greater than about 95%, greater than about 97%, or greaterthan about 99% of the monooxygenase produced by a culture. In otherembodiments, essentially only shall refer to the ratio of production ofpMMO to sMMO. In some embodiments wherein a culture is producingessentially only pMMO, the ration of pMMO to sMMO is about 2:1, about5:1, about 10:1 about 50:1, about 100:1, about 250:1, about 500:1, about1000:1, about 2500:1, about 5000:1, about 10,000:1, or greater. In stilladditional embodiments, the culture may produce both sMMO and pMMO, butwith respect to the activity of the enzymes, the culture produces PHAessentially only through the pMMO-mediated pathway. In one embodiment,therefore, even if the culture comprises an equal concentration of sMMOand pMMO, PHA may be preferentially produced via pMMO. In otherembodiments, depending on the culture conditions and/or themicroorganism strain, the inverse can occur (e.g., the culture producesessentially only sMMO). In one embodiment, the concentration of sMMO inthe culture is more than 2 times greater than the concentration of pMMOin the culture. In one embodiment, the concentration of sMMO is morethan 5 times greater than the concentration of pMMO in the culture. Inone embodiment, the concentration of sMMO is more than 10 times greaterthan the concentration of pMMO in culture. In one embodiment, theconcentration of pMMO is more than 2 times greater than theconcentration of sMMO in said culture. In one embodiment, theconcentration of pMMO is more than 5 times greater than theconcentration of sMMO in said culture. In one embodiment, steps (a)through (c) are repeated, wherein the concentration of sMMO relative topMMO in the culture is substantially the same (e.g., within 5%, 10%,25%, 50% or 75% relative proportion by weight) in step (c) in at leasttwo repetitions.

In one embodiment, a method is provided for converting acarbon-containing gas or material into a polyhydroxyalkanoate (PHA) athigh efficiency, comprising: (a) providing a methanotrophic culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture wherein the gene encoding the soluble methane monooxygenaseenzyme is absent in the one or more microorganisms, (d) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d). Copperis a critical component of many methanotrophic systems, including bothsMMO and pMMO, and copper typically controls the switch between sMMO andpMMO production. Reducing copper concentration (below 0.1, 1, 2, 4, 10,20, 40, 100 micromolar, below 0.001, 0.01, 0.1, 1, 2, 4, 8, 10, 15, 20,40, 100, 200 mg/L, or below 0.001, 0.01, 0.1, 1, 2, 4, 10, 100 mg/g dryweight of microorganism biomass) typically increases sMMO production,while increasing copper concentration (above 0.1, 1, 2, 4, 10, 20, 40,100 micromolar, above 0.001, 0.01, 0.1, 1, 2, 4, 8, 10, 15, 20, 40, 100,200 mg/L, or, or above 0.001, 0.01, 0.1, 1, 2, 4, 10, 100 mg/g dryweight of microorganism biomass) typically increases pMMO production.Copper is generally added to a culture each time water or mineral mediais added to a culture, since trace copper is difficult to remove fromeven purified water, and copper is needed for methanotrophic cellularreplication/growth, since MMO generally drives the oxidation of methaneto biomass, and MMO is a copper-containing enzyme. Applicant hassurprisingly discovered that microorganisms that do not possess orexpress the gene for sMMO unexpectedly produce PHA at high efficiency,and can be selectively cultured, using culture selection pressures, toout-compete microorganisms that do possess or express the gene for sMMO(particularly in non-sterile systems, wherein new microorganisms areperiodically introduced to the culture) by limiting, controlling, orreducing copper concentrations and simultaneously subjecting the cultureto growth-polymerization-growth repetitions, as described herein. In oneembodiment, microorganisms that possess higher concentrations of PHAswitch from polymerization to growth mode (wherein the microorganismsproduce soluble and/or particulate methane monooxygenase) more quicklyand efficiently than microorganisms that possess lower concentrations ofPHA, and/or carry out cellular replication more efficiently in general;by reducing copper concentrations to levels that would traditionallycause the culture to express sMMO (e.g., less than 0.001, 0.01, 0.1, 1,10, or 100 mg/L, or less than 0.01, 0.1, 1, 10, or 100 micromolar, orless than 0.001, 0.01, 0.1, 1, 10, or 100 mg per gram microorganism dryweight) while also cycling between growth and polymerization cycles,microorganisms that produce high concentrations of PHA and also growquickly in low copper concentrations out-compete microorganisms thatproduce less PHA and grow slower in low copper concentrations. SincepMMO renders a faster metabolism than sMMO, by reducing the copperconcentration in the medium (permanently or temporarily) totraditionally sMMO-generating concentrations, and concurrentlysubjecting a culture to growth-polymerization-growth repetitions whichselect for microorganisms that generate high PHA concentrations andmetabolize efficiently in transitioning from polymerization mode togrowth mode, Applicant has discovered that a high-efficiencymicroorganism can be selectively produced and maintained, including innon-sterile conditions, that does not contain or express the geneticcoding for sMMO, accumulates high concentrations of PHA, andout-competes microorganisms that produce sMMO. The ability to cause amethanotrophic culture to generate microorganisms that do not possess orexpress the gene encoding sMMO by reducing copper concentrations is asurprising and unexpected result, and offers a range of advantages,including superior process stability (microorganisms produce only pMMO,regardless of copper concentration), PHA consistency (the metabolicpathway, pMMO, is unchanging, thereby controlling the characteristics ofPHA produced, such as monomer composition, molecular weight,polydispersity, elongation, modulus, viscosity), and increased metabolicefficiency (e.g., rate, oxidation, metabolism, copper requirements). Inaddition to copper, other nutrients can be used, either individually orin combination, in similar fashion to control for the selectiveproduction of microorganisms that do not possess or express the geneencoding soluble methane monooxygenase, such nutrients including:methane, oxygen, phosphorus, magnesium, iron, boron, aluminum, calcium,cobalt, chloride, chromium, EDTA, manganese, molybdenum, sulfur, nickel,zinc, and/or potassium. In one embodiment, more than 10%, 25%, 50%, 75%,or 80% of the culture does not contain or express the gene encodingsoluble methane monooxygenase.

In one embodiment, a method is provided for converting acarbon-containing material (e.g., methane, carbon dioxide, propane,ethane, acetone, acetate, formaldehyde, a volatile organic compound, anon-methane organic compound, carbon dioxide) into apolyhydroxyalkanoate (PHA), the method comprising: (a) providing amethanotrophic culture, (b) providing a medium comprising one or morenutrient comprising a carbon-containing material that can be metabolizedby the culture, (c) controlling the concentration of the one or morenutrient in the medium to cause the cellular replication of the culturew wherein the gene encoding the ethylmalonyl-CoA pathway is present orexpressed in one or more microorganism in said culture, (d) controllingthe concentration of said one or more nutrients in said medium to causesaid culture to produce PHA, and (e) repeating steps (a) through (d).Applicant has surprisingly discovered that the ethylmalonyl-CoA (EMC)pathway, combined with required and controlled conditions, enables theproduction of over 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, and 95%intracellular PHA concentrations. Applicant has also surprisinglydiscovered that the ethylmalonyl-CoA (EMC) pathway can switch fromgrowth to PHA polymerization and back to growth at high efficiency, evenwhen intracellular PHA concentrations exceed over 50%, 55%, 60%, 65%,70%, 80%, 85%, 90%, and 95% by weight, whereas many cultures with PHAconcentrations exceeding 50%, 60%, or 70% cannot effectively return togrowth mode following a polymerization period. Thus, by subjecting aculture to growth-polymerization-growth cycling, Applicant has foundthat it is possible to select for microorganisms that possess the geneencoding the EMC pathway. In addition, Applicant has surprisinglydiscovered that it is possible to selectively produce a culture ofmicroorganisms that express the EMC pathway in sterile or non-sterileconditions, including in the presence of methane, non-methanecarbon-containing materials, or other materials that influence themetabolism of the microorganisms, by simultaneously subjecting theculture to growth-PHA polymerization-growth cycling, as describedherein, while also controlling the concentration one or more nutrientsavailable to the culture. In one embodiment, the concentration of anutrient, such as copper, is controlled to selectively favor theproduction of EMC-pathway microorganisms. In some embodiments,microorganisms that do not possess or express the genetic materialencoding soluble methane monooxygenase also possess the genes encodingthe EMC-pathway. Thus, as described herein, by subjecting the culture togrowth-polymerization-growth (GPG) cycling while also controlling orreducing the concentration of copper, or other nutrients that impact theswitch between sMMO and pMMO production, it is possible to selectivelyproduce microorganisms that do not possess or express the geneticmaterial encoding soluble methane monooxygenase but do possess the geneencoding the EMC pathway. The ability to selectively producemicroorganisms that contain or express the genes encoding the EMCpathway and/or do not contain or express the genes encoding sMMO offerssignificant process advantages, including process stability(microorganisms produce consistent concentrations of PHA and produceonly pMMO, regardless of copper concentration or microorganismcontamination), PHA consistency (the metabolic pathway is unchanging,thereby controlling the characteristics of PHA produced, such as monomercomposition, molecular weight, polydispersity, elongation, modulus,viscosity), and increased metabolic efficiency (e.g., rate, oxidation,metabolism, nutrient requirements). In some embodiments, suchmicroorganisms and processes may be combined with other microorganismsand processes, wherein a culture may contain microorganisms containinggenes encoding or expressing both sMMO and pMMO and genetic packagesthat do not encode or express the EMC pathway; in some embodiments,microorganisms that do not possess or express one or more gene encodingsMMO comprise less than 1%, less than 5%, less than 10%, less than 20%,less than 50%, less than 75%, more than 75% of a culture; in otherembodiments, microorganisms that contain or express genetic materialencoding the EMC pathway comprise less than 1%, less than 5%, less than10%, less than 20%, less than 50%, less than 75%, more than 75% of aculture.

In one embodiment, a method is provided for converting acarbon-containing gas or material into a polyhydroxyalkanoate (PHA) athigh efficiency, comprising: (a) providing a methanotrophic culture, (b)providing a medium comprising one or more nutrient comprising acarbon-containing material that can be metabolized by the culture, (c)controlling the concentration of the one or more nutrient in the mediumto cause the cellular replication of one or more microorganisms in theculture wherein the gene encoding the soluble methane monooxygenaseenzyme is absent in the one or more microorganisms, (d) controlling theconcentration of the one or more nutrients in the medium to cause theculture to produce PHA, and (e) repeating steps (a) through (d). Copperis a critical component of many methanotrophic systems, including bothsMMO and pMMO, and copper typically controls the switch between sMMO andpMMO production. In several embodiments, reducing copper concentration(below about 0.1, about 1, about 2, about 4, about 10, about 20, about40, about 100 micromolar, below about 0.001, about 0.01, about 0.1,about 1, about 2, about 4, about 8, about 10, about 15, about 20, about40, about 100, about 200 mg/L, or below about 0.001, about 0.01, about0.1, about 1, about 2, about 4, about 10, about 100 mg/g dry weight ofmicroorganism biomass) typically increases sMMO production. In contrast,increasing copper concentration (above about 0.1, about 1, about 2,about 4, about 10, about 20, about 40, about 100 micromolar, above about0.001, about 0.01, about 0.1, about 1, about 2, about 4, about 8, about10, about 15, about 20, about 40, about 100, about 200 mg/L, or, orabove about 0.001, about 0.01, about 0.1, about 1, about 2, about 4,about 10, about 100 mg/g dry weight of microorganism biomass) typicallyincreases pMMO production. Copper is generally added to a culture eachtime water or mineral media is added to a culture, since trace copper isdifficult to remove from even purified water, and copper is needed formethanotrophic cellular replication/growth, since MMO generally drivesthe oxidation of methane to biomass, and MMO is a copper-containingenzyme.

In accordance with several embodiments, Applicant has surprisinglydiscovered that selective culture conditions can be employed that allowfor the dominance of a culture by microorganisms that utilize the pMMOpathway, even at low or reduced copper concentrations. In someembodiments, the pMMO pathway or enzyme is the exclusive pathway orenzyme expressed or used in the microorganism for the production ofmethane monooxygenase (e.g., the selective pressures of the processesdisclosed herein induce a loss of the genetic material encoding the sMMOgene in the culture or microorganism over time, or the selectivepressures result in the dominance or growth or metabolic success ofmicroorganisms that do not possess or express the genetic material usedfor sMMO production). In some embodiments, the pMMO pathway is eitherpreferentially expressed or preferentially active in the culture, suchthat the microorganisms still retain the genetic material necessary toproduce sMMO, but sMMO is either not produced, produced but not used,produced and functionally blocked (e.g., negative feedback mechanisms),produced and functionally deficient under the culture conditions, and/orproduced but metabolically outcompeted for substrate by pMMO enzymes.Thus, in several embodiments, microorganisms that have reduced sMMOexpression or function unexpectedly produce PHA at high efficiency, andcan be selectively cultured, using culture selection pressures, toout-compete microorganisms that do express sMMO (particularly innon-sterile systems, wherein new microorganisms are periodicallyintroduced to the culture) by limiting, controlling, or reducing copperconcentrations and simultaneously subjecting the culture togrowth-polymerization-growth repetitions, as described herein.

In one embodiment, microorganisms that possess higher concentrations ofPHA switch from polymerization to growth mode (wherein themicroorganisms produce soluble and/or particulate methane monooxygenase)more quickly and efficiently than microorganisms that possess lowerconcentrations of PHA, and/or carry out cellular replication moreefficiently in general. By reducing copper concentrations to levels thatwould cause or enable, or traditionally cause or enable, the culture, orat least one or more methanotrophic microorganisms, to express sMMO(e.g., less than about 0.001, about 0.01, about 0.1, about 1, about 10,or about 100 mg/L, or less than about 0.01, about 0.1, about 1, about10, or about 100 micromolar, or less than about 0.001, about 0.01, about0.1, about 1, about 10, or about 100 mg per gram microorganism dryweight) while also cycling between growth and polymerization cycles,microorganisms that produce high concentrations of PHA and also growquickly in low copper concentrations out-compete microorganisms thatproduce less PHA and grow slower in low copper concentrations. SincepMMO renders a faster metabolism than sMMO, by reducing the copperconcentration in the medium (permanently or temporarily) as compared totraditionally pMMO-generating copper concentrations (e.g., toconcentrations that could enable or induce one or more methanotrophicmicroorganisms to produce sMMO if present in the medium), andconcurrently (or subsequently) subjecting a culture togrowth-polymerization-growth repetitions which select for microorganismsthat generate high PHA concentrations and metabolize efficiently intransitioning from polymerization mode to growth mode, Applicant hasdiscovered that a high-efficiency microorganism can be selectivelyproduced and maintained (even in non-sterile conditions) that eitherdoes not contain, does not express, does not produce, or expresses orproduces at reduced levels sMMO, accumulates high concentrations of PHA,and out-competes microorganisms that produce sMMO. The ability to causea methanotrophic culture to generate microorganisms that do not possessthe genetic material encoding sMMO or express sMMO at reduced levels (orreduced functionality) by reducing copper concentrations is a surprisingand unexpected result, and offers a range of advantages. For example,superior process stability (microorganisms produce only pMMO, regardlessof copper concentration), PHA consistency (the metabolic pathway, pMMO,is unchanging, thereby controlling the characteristics of PHA produced,such as monomer composition, molecular weight, polydispersity,elongation, modulus, viscosity), and increased metabolic efficiency(e.g., rate, oxidation, metabolism, copper requirements) are achieved.In addition to copper, other nutrients can be used, either individuallyor in combination, in similar fashion to control for the selectiveproduction of microorganisms that do not possess or express the geneencoding soluble methane monooxygenase, such nutrients including:methane, oxygen, phosphorus, magnesium, iron, boron, aluminum, calcium,cobalt, chloride, chromium, EDTA, manganese, molybdenum, sulfur, nickel,zinc, and/or potassium. In one embodiment, more than 10%, 25%, 50%, 75%,or 80% of the culture does not contain the gene encoding soluble methanemonooxygenase.

In one embodiment, a method is provided for converting acarbon-containing material (e.g., methane, carbon dioxide, propane,ethane, acetone, acetate, formaldehyde, a volatile organic compound, anon-methane organic compound, carbon dioxide) into apolyhydroxyalkanoate (PHA), the method comprising: (a) providing amethanotrophic culture, (b) providing a medium comprising one or morenutrient comprising a carbon-containing material that can be metabolizedby the culture, (c) controlling the concentration of the one or morenutrient in the medium to cause the cellular replication of the culturewherein the genetic material encoding the ethylmalonyl-CoA (EMC) pathway(e.g., the various enzymes or co-factors that are involved in thepathway) is present in one or more microorganism in said culture, (d)controlling the concentration of said one or more nutrients in saidmedium to cause said culture to produce PHA, and (e) repeating steps (a)through (d). Applicant has surprisingly discovered that the EMC pathway,combined with required and controlled conditions, enables the productionof over 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, and 95% intracellularPHA concentrations. Applicant has also surprisingly discovered that themicroorganisms using the EMC pathway can be switched from growthconditions or metabolism to PHA polymerization conditions or metabolismand back to growth conditions or metabolism at high efficiency, evenwhen intracellular PHA concentrations exceed over 50%, 55%, 60%, 65%,70%, 80%, 85%, 90%, and 95% by weight, whereas many cultures with PHAconcentrations exceeding 50%, 60%, or 70% cannot effectively return togrowth mode following a polymerization period. Thus, by subjecting aculture to growth-polymerization-growth cycling, Applicant has foundthat it is possible to select for microorganisms that possess the geneencoding the EMC pathway. In addition, Applicant has surprisinglydiscovered that it is possible to selectively produce a culture ofmicroorganisms that express the EMC pathway in sterile or non-sterileconditions, including in the presence of methane, non-methanecarbon-containing materials, or other materials that influence themetabolism of the microorganisms, by simultaneously subjecting theculture to growth-PHA polymerization-growth cycling, as describedherein, while also controlling the concentration one or more nutrientsavailable to the culture. In one embodiment, the concentration of anutrient, such as copper, is controlled to selectively favor theproduction of EMC-pathway microorganisms. In some embodiments,microorganisms that do not possess the genetic material encoding solublemethane monooxygenase (or have the genetic material but express atreduced levels or at reduced levels of activity) also possess the genesencoding the EMC-pathway. Thus, as described herein, by subjecting theculture to growth-polymerization-growth (GPG) cycling while alsocontrolling or reducing the concentration of copper, or other nutrientsthat impact the switch between sMMO and pMMO production, it is possibleto selectively produce microorganisms that do not possess the geneencoding soluble methane monooxygenase (or have the genetic material butexpress sMMO at reduced levels or at reduced levels of activity) but dopossess the gene encoding the EMC pathway. The ability to selectivelyproduce microorganisms that contain the genetic material for and expressthe gene encoding the EMC pathway and/or do not contain the geneticmaterial for or do not express the gene encoding sMMO offers significantprocess advantages, including process stability (microorganisms produceconsistent concentrations of PHA and produce only pMMO, regardless ofcopper concentration or microorganism contamination), PHA consistency(the metabolic pathway, is unchanging, thereby controlling thecharacteristics of PHA produced, such as monomer composition, molecularweight, polydispersity, elongation, modulus, viscosity), and increasedmetabolic efficiency (e.g., rate, oxidation, metabolism, nutrientrequirements). In some embodiments, such microorganisms and processesmay be combined with other microorganisms and processes, wherein aculture may contain microorganisms containing genes encoding both sMMOand pMMO and genetic material that does not include material encodingthe EMC pathway; in some embodiments, microorganisms that do not possessgene(s) encoding sMMO comprise less than about 1%, less than about 5%,less than about 10%, less than about 20%, less than about 50%, less thanabout 75%. In other embodiments, microorganisms that contain geneticmaterial encoding the EMC pathway comprise less than about 1%, less thanabout 5%, less than about 10%, less than about 20%, less than about 50%,or less than about 75%. In some embodiments, microorganisms andprocesses may be combined with other microorganisms and processes,wherein a culture may contain microorganisms containing genes encodingboth sMMO and pMMO and genetic material that does not include materialencoding the EMC pathway. It shall be appreciated that additionalselection methods, and/or “spiking” of a culture with microorganisms ofa certain type or genetic makeup can be used to achieve microorganismcultures with desired characteristics/demographics. [0146] In severalembodiments of the invention, methanol is added to a culture ofmethanotrophic microorganisms utilizing a closed loop recycling gasstream comprising methane. In some embodiments, methanotrophicmicroorganisms are enabled to grow under conditions of, and consume,very low concentrations of methane by co-utilizing methanol as a carbonsubstrate. In the past, the growth of methanotrophic microorganisms wassignificantly reduced under low methane concentrations due to, amongother things, low mass transfer rates. In some embodiments, by theaddition of methanol in a closed loop gas recycling system, it ispossible to effect the substantially complete elimination of methane bymethanotrophic microorganisms.

In several embodiments of the invention, the diffusion of light isincreased in a liquid growth culture media by reducing the density ofthe liquid in a light path. In some embodiments the culture comprisesautotrophic microorganisms. In some embodiments, the application of gasbubbles into the media decreases the relative solids density of thelight path, thus enabling an increased diffusion of light into a liquidculture media from a given light intensity energy.

In several embodiments of the invention, a series of submerged lightrods are placed into a liquid culture to manipulate or adjust theculture conditions. In some embodiments, the culture comprisesautotrophic microorganisms. In some embodiments, the light rods functionto diffuse light, diffuse gas, act as static or dynamic mixers, assistin the circulation of a liquid culture media, and/or facilitate heatexchange through the circulation of a gas, liquid, and/or combinationthereof.

Traditionally, pH control in a microorganism growth system is difficultand/or costly to maintain. In some embodiments, pH is controlled byvarying the nitrogen source supplied to a microorganism growth systembetween pH-increasing and pH-reducing nitrogen sources, e.g., NO₃ ⁻ andNH₃ ⁺, respectively. In some embodiments, nitrogen sources are utilizedthat do not significantly affect the pH of the system, including, whenapplicable, complex nitrogen sources such as biomass. In additionalembodiments, a closed loop system is employed to reduce changes in pH.In some embodiments, respiration-generated carbon dioxidecounterbalances increases in pH caused by the utilization ofpH-increasing nitrogen sources, such as nitrates. In one embodiment,nitrogen fixation is used to add hydroxyl ions to the culture medium,which may or may not be counterbalanced by the addition of protons fromeither biological or chemical sources. In one embodiment, nitratefixation is used to add hydroxyl ions to the culture medium, which mayor may not be counterbalanced by the addition of protons from eitherbiological or chemical sources. In one embodiment, ammonia or ammoniumfixation is used to add protons to the culture medium, which may or maynot be counterbalanced by the addition of hydroxyl ions from eitherbiological or chemical sources.

A number of methods are known for the induction of gas into liquid,including static mixing, ejector mixing, propeller mixing, and/or acombination thereof. Simultaneously, it is also known that shear can behighly detrimental to microorganism growth, and can often impede orpermanently deactivate microorganism metabolism. Thus, mass transfer ina gas-based system is often limited by the need to counterbalancesufficient mixing with shear considerations. In several embodiments ofthe invention, a vessel comprising liquid culture media is mixed with agas, e.g. methane, under relatively high shear conditions, and thensubsequently transferred to a vessel comprising liquid culture mediamaintained under relatively low shear conditions. In some embodiments,microorganism growth is primarily induced in the low shear vessel. Insome embodiments, high gas transfer rates are effected in the first highshear vessel by mixing while performed in the low shear vessel bygaseous diffusion.

In another embodiment, a closed loop gas recycling system is maintained,wherein a vessel comprising gas-utilizing microorganisms is suppliedwith gas, wherein the gas is utilized by gas-utilizing microorganisms,and wherein the rate at which gas is added to the system is determinedby the rate at which the pressure in the vessel changes in accordancewith the conversion of gases into metabolic derivatives (such asbiomass, carbon dioxide, and water). For example, a vessel containingmethane-utilizing microorganisms may be pressurized to 60 psi with acombination of methane and oxygen; as the pressure in the system dropsin accordance with the metabolism of the methane-utilizingmicroorganisms, additional methane and oxygen is added to the systemsuch that the pressure of the vessel remains at 60 psi. It shall beappreciated that, in certain embodiments, higher or lower pressures aremaintained. In some embodiments, the system is periodically flushed toremove carbon dioxide. In some embodiments, autotrophic microorganismsand a light injection system may be added to the system in order toconvert carbon dioxide into additional oxygen, thereby substantiallyreducing or eliminating the need to flush the system and/or introduceoxygen.

In several embodiments, PHA synthesis is induced in a microorganismculture comprising methane-utilizing, heterotrophic, and/or carbondioxide-utilizing microorganisms wherein a PHA inclusion concentration(by dry biomass weight) is generated of between about 0.01% and about95%. In some embodiments, the inclusion concentration is between about25% and about 80%, including about 25 to about 35%, about 35% to about50%, about 50% to about 65%, about 65% to about 80%, and overlappingranges thereof. In some embodiments, the inclusion concentration isbetween about 0.01% and about 55%, including, about 0.01% to about 1%,about 1% to about 5%, about 5% to about 10%, about 10% to about 15%,about 15% to about 20%, about 20%, to about 25%, about 25% to about 30%,about 30% to about 35%, about 35% to about 40%, about 40% to about 45%,about 45% to about 50%, about 50% to about 55%, and overlapping rangesthereof. In some embodiments, these inclusion ratios are achieved withreduced carbon input, reduced energy expenditure, or reduced numbers ofmicroorganisms (thereby representing a more efficient generation ofPHA). In some embodiments, PHA synthesis is induced in a methanotrophic,heterotrophic, and/or autotrophic microorganism culture wherein a PHAinclusion concentration (by dry biomass weight) is generated of betweenabout 20% and about 80%, between about 30% and about 70%, between about40% and about 60%, between about 50% and about 70%, including about 50%to about 55%, about 55 to about 60%, about 60% to about 65%, about 65%to about 70%, and overlapping ranges thereof. In some embodiments of theinvention, PHA synthesis is induced in microorganism culture comprisingmethanotrophic, autotrophic, and heterotrophic microorganisms, whereinan average PHA inclusion concentration (by dry biomass weight) isgreater than about 5%, greater than about 20%, greater than about 40%,greater than about 65%, or greater than about 70% by dry cell weight.

In some embodiments, the growth culture media is manipulated to induceboth i) microorganism growth and ii) PHA synthesis within one or moreopen, non-sterile, or sterile vessels using a feast-famine cultureregime. In some embodiments, the microorganisms are subject tosuccessive alternating periods of nutrient/carbon availability andnutrient/carbon unavailability to encourage the reproductive success ofmicroorganisms that are capable of synthesizing PHA, particularly athigh inclusion concentrations. Feast-famine regimes useful for theselection of PHA producing microorganisms, including PHA-producingmethanotrophic microorganisms, over microorganisms that either cannotproduce PHA, produce PHA slowly, or produce PHA at relatively lowconcentrations are described in the art (Verlinden, et al., “Bacterialsynthesis of biodegradable polyhydroxalkanoates,” Journal of AppliedMicrobiology, 102 (2007), p. 1437-1449, Frigon, et al., “rRNA andPoly-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast andFamine Cycles,” Applied and Environmental Microbiology, April 2006, p.2322-2330; Müller, et al., “Adaptive responses of Ralstonia eutropha tofeast and famine conditions analysed by flow cytometry,” J Biotechnol.1999 Oct. 8; 75(2-3):81-97; Reis, et al., “Production ofpolyhydroxyalkanoates by mixed microbial cultures,” Bioprocess andBiosystems Engineering, Volume 25, Number 6, 377-385, DOI:10.1007/s00449-003-0322-4.)

In some embodiments of the invention, the classical feast-famine regimeis modified to reduce PHA losses. Specifically, in the past,feast-famine regimes were thought to be effective by passing amicroorganism culture through a period wherein carbon or nutrients wereunavailable or relatively limited for metabolism, thereby forcing theculture to accumulate and/or consume intracellular PHA as a source ofcarbon to survive, and thereby selecting for microorganisms with thecapacity to synthesize and store PHA. Applicant has surprisinglydiscovered that, some microorganisms with higher concentrations ofintracellular PHA reproduce more efficiently than microorganisms withlower concentrations of intracellular PHA in periods of carbonavailability and nutrient balance. Thus, in one embodiment, a novel PHAproduction regime is employed in one or more vessel whereinmicroorganisms are subjected to two successive and recurring phases: 1)growth, wherein carbon and nutrient availability is optimized forreproduction, and 2) PHA synthesis, wherein carbon is available inexcess, and one or more nutrient is reduced or increased relative to thegrowth period to induce PHA synthesis. In some embodiments, a fractionof the vessel media is removed for downstream PHA extraction andprocessing after the PHA synthesis period, and that fraction is replacedwith lower cell density media, which simultaneously returns carbon andnutrient concentrations to reproductively favorable levels, e.g., thegrowth phase or growth conditions, and causes microorganisms to enterinto a reproductive phase without consuming significant portions ofintracellular PHA. As such, efficient PHA producing microorganismsselectively reproduce over inefficient or non-PHA producingmicroorganisms. As a result, some embodiments, i) increase the speed ofthe microorganism selection process by removing the PHA consumption steptypical to previous feast famine models and ii) reduce the loss of PHAto cellular metabolism. According to such embodiments, the feast faminemodel is converted to a feast-polymerization-feast process. In severalembodiments methanotrophic, autotrophic, and/or heterotrophic culturesare used in the feast-polymerization-feast process.

Removing a Portion of the PHA-Containing Biomass from the Culture, andExtracting PHA from the Removed PHA-Containing Biomass to ProduceIsolated PHA and PHA-Reduced Biomass

In several embodiments, following the production of a microorganismculture comprising biomass and PHA (discussed above), at least a portionof the PHA-containing biomass is removed from the culture. In severalembodiments, a portion ranging from about 20% to about 80% of thePHA-containing biomass is removed, including about 30% to about 70%,about 40% to about 60%, about 45% to about 55%, and overlapping rangesthereof. Removal of PHA-containing biomass may be performed by a numberof methods, including centrifugation, filtration, density separation,flocculation, agglomeration, spray drying, or other separationtechnique. In some embodiments, dewatering (e.g., by centrifugation)results in a biomass having a desirable water content that facilitatesdownstream processing of the biomass. For example, in some embodiments,centrifugation of the PHA-containing biomass reduces the amount ofculture media (increases the relative biomass concentration) to aconcentration range between about 100 and about 500 grams of biomass perliter of culture media. In some embodiments, the concentration is of thebiomass is adjusted to about 100 to about 200 g/L, about 200 to about300 g/L, about 300 to about 400 g/L, about 400 to about 500 g/L, andoverlapping ranges thereof. Advantageously, such an approach alsoproduces, as an effective by-product, clarified culture media that canbe optionally treated, measured, or recycled into one or more culturevessels.

In some embodiments, after a portion of the PHA-containing biomass isremoved from the culture, PHA is extracted from the removedPHA-containing biomass to produce isolated PHA and PHA-reduced biomass.

As used herein, the terms “extraction” and “PHA extraction” shall begiven their ordinary meaning and shall be used interchangeably todescribe the removal and/or separation of PHA from biomass. PHAs may beextracted from biomass by several processes, including, but not limitedto, the use of chemicals (e.g., solvents) alone or in combination withmechanical means and/or enzymes. These processes include the use of:solvents, such as acetone, ethanol, methanol, methylene chloride,dichloroethane, with and/or without the application of pressure and/orelevated temperatures, supercritical carbon dioxide, enzymes, such asproteases, surfactants, pH adjustment, including the protonic orhydroxide-based dissolution of non-PHA biomass, and/or hypochlorite (oranother solvent) to dissolve non-PHA biomass, including the use ofhypochlorite in conjunction with another solvent, such as methylenechloride or with, carbon dioxide, enzymes, acids, bases, polymers, orsurfactants, or combinations thereof. In some embodiments of theinvention, PHA is extracted by solvent extraction from a PHA-containingbiomass comprising gas-utilizing microorganisms and/or biomass-utilizingmicroorganisms to produce isolated PHA and PHA-reduced biomass. In somesolvent-based embodiments, solvents suitable for dissolving the PHA areused, including carbon dioxide, acetone, methylene chloride, chloroform,water, ethanol, and methanol. In some embodiments, particular ratios ofsolvent to PHA provide optimal dissolution of PHA from the culture, andtherefore lead to improved extraction and isolation efficiency andyield. For example, in some embodiments, ratios of solvent to PHA (ingrams) of about 500:1 are used. In some embodiments, ratios of about0.01:1 are used. In some embodiments, ratios ranging from between about500:1 and about 0.01:1 are used, such as about 0.05:1, about 1.0:1,about 1.5:1, about 20:1, about 250:1, about 300:1, about 350:1, about400:1, or about 450:1.

As discussed above, changes in temperature and/or pressure may also beused to facilitate the extraction of PHA from the PHA-containingbiomass. In some embodiments, the extraction solvent chosen dictates thelimits of temperature, pressure, and/or incubation times that are used.In some embodiments, solvent is combined with PHA-containing biomass andincubated for several minutes up to several hours. For example, in someembodiments, incubation is for about 10 minutes, while in otherembodiments, overnight incubation times are used. In some embodiments,incubation times range from 30 minutes to about 1 hour, about 1 hour toabout 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours,and from about 10 hours to overnight. Choice of incubation time isdetermined by solvent, culture density (e.g., number of microorganisms),type of organisms, expected PHA yield, and other similar factors.

Incubation temperature is also tailored to the characteristics of agiven culture. Incubation temperatures can range from below roomtemperature to elevated temperatures of up to about 150° C. or about200° C. For example, depending the solvent and other variables of theculture, temperatures are used that range from about 10° C. to 25° C.,from about 25° C. to about 40° C., from about 40° C. to about 55° C.,from about 55° C. to about 60° C., from about 60° C. to about 75° C.,from about 75° C. to about 90° C., from about 90° C. to about 105° C.,from about 105° C. to about 120° C., from about 120° C. to about 135°C., from about 135° C. to about 150° C., from about 150° C. to about200° C., and overlapping ranges thereof.

As can be appreciated, if changes in temperature are made to a culturein a closed vessel, changes in pressure result. In some embodiments,increased pressure provides a shearing effect that aids in theliberation of PHA from the microorganisms. In some embodiments, pressureis regulated within a particular range. For example, in someembodiments, pressure of the reaction of the PHA-containing biomass withsolvent occurs between about 40 and 30,000 psi, including about 50 toabout 60 psi, about 60 to about 70 psi, about 70 to about 80 psi, about80 to about 90 psi, about 90 to about 100 psi, about 100 to about 125psi, about 125 to about 150 psi, about 150 to about 175 psi, about 175to about 200 psi, about 200 to about 1000 psi, about 1000 to about 5000psi, about 5000 psi to about 10,000 psi, about 10,000 to about 20,000psi, about 20,000 to about 30,000 psi, and overlapping ranges thereof.Additional sources of shear (e.g., agitation, pumping, stirring etc.)are optionally used in some embodiments to enhance the extraction ofPHA. Any one, or combination, of the PHA extraction methods describedherein, or disclosed in the art, may be utilized as a method to carryout PHA extraction and remove PHA from the PHA-containing biomass.

In several embodiments, a solvent-based extraction system is utilized tocarry out PHA extraction. In some embodiments, solvents are utilized tocarry out PHA extraction at high temperatures, wherein PHA extractionoccurs simultaneous with a temperature-enhanced breakdown or dissolutionof PHA-containing biomass. In some embodiments, one or more solvent isutilized that is biodegradable and metabolically assimilable by theculture, such that PHA-reduced biomass comprising biomass and one ormore biodegradable solvent may be contacted with the culture, and boththe PHA-reduced biomass and the solvent may be utilized by the cultureas a source of carbon. Non-limiting examples of such solvents includecarbon dioxide, acetone, ethanol, methanol, and methylene chloride,among others.

In several embodiments a mixture of solvent and PHA comprises multiplephases, e.g. an aqueous phase and an organic phase. In some embodiments,solvent-based extraction comprises a more uniform mixture of solvent andPHA. In some embodiments, depending on the solvent the phases areseparated prior to recovery of the PHA. In some embodiments, a non-PHApolymer is also used, alone or in conjunction with other processes, forseparation, flocculation, or other processing. In some embodiments,centrifugation is employed to further distinguish and separate thephases of the mixture (e.g., separation of the solvent-PHA phase fromthe water-biomass phase). In some embodiments, heat is also employed tomaintain the solubility of the PHA in a given solvent.

It shall be appreciated that the solubility of PHA varies with thesolvent used, and therefore the temperature (if adjusted) and theseparation techniques are tailored to match the characteristics of agiven solvent. Thus, in some embodiments employing centrifugation, forexample, a low speed centrifugation is used to separate the solvent-PHAphase from the water-biomass phase. In other embodiments, depending onthe solvent, higher speed centrifugation is used. In some embodiments,centrifugation is employed in stages, e.g., low speed centrifugationfollowed by high speed centrifugation. Any of a variety of centrifugescan be employed, depending on the solvent used, for example, basketcentrifuges, swinging bucket centrifuges, fixed rotor centrifuges,disc-back centrifuges, supercentrifuges, or ultracentrifuges.

In some embodiments, adjustable discharge ports suitable for aparticular centrifuge are used in order to control the rate and degreeof separation of solvent-PHA phase from the water-biomass phase. In someembodiments, the concentration of water in the water-biomass phase isadjusted to allow for suitable flow of the mixture through thecentrifuge (or within a centrifuge tube). For example, in someembodiments, flow is suitable for separating the phases when theconcentration of biomass (relative to water) is between about 1 and 100g/L. In some embodiments, the concentration is between about 10 to about20 g/L, about 20 to about 30 g/L, about 30 to about 40 g/L, about 40 toabout 50 g/L, about 50 to about 60 g/L, about 60 to about 70 g/L, about70 to about 80 g/L, about 80 to about 90 g/L, about 90 to about 100 g/L,about 100 to about 200 g/L, about 200 to about 400 g/L, about 400 toabout 600 g/L, and overlapping ranges thereof.

In still additional embodiments, increases in temperature not onlyfacilitate the extraction of the PHA, they also facilitate the isolationof the PHA from the solvent (e.g. increased temperature increasessolvent evaporation).

In some embodiments, an extraction process is carried out to remove PHAfrom a microorganism in such a manner that the microorganism isdeactivated. In some embodiments, the deactivation is permanent, whilein some embodiments the deactivation is temporary. Without being boundby theory, it is believed that PHA extraction techniques which do notpermanently disable microorganisms enable the PHA-reduced biomassgenerated thereby to contribute to the metabolism of carbon sourcesafter a PHA extraction process, including through intracellular andextracellular metabolism. In one embodiment, methods useful for thetemporary disablement of microorganisms include solvent extraction,including solvent extraction carried out below about 100° C., andparticularly at intracellular temperatures below about 100° C.,including extraction temperatures of about 10° C. to about 30° C., about30° C. to about 50° C., about 50° C. to about 60° C., about 60° C. toabout 70° C., about 70° C. to about 80° C., about 80° C. to about 90°C., about 90° C. to about 100° C., and overlapping ranges thereof.

In several embodiments, the PHA concentration of PHA-containing biomassis reduced as a result of the PHA extraction process. In severalembodiments, the PHA concentration of PHA-containing biomass is reducedby at least about 0.01% (by dry cell weight). In some embodiments, thePHA concentration is reduced by about 10% to about 50%, about 50% toabout 70%, about 70% to about 75%, about 75% to about 80%, about 80% toabout 85%, about 85%, to about 90%, about 90% to about 95%, about 95% toabout 99.9%, and overlapping ranges thereof.

While a variety of methods are known to enable the extraction PHA frombiomass, most methods can be categorized into one of two classes: a)solvent-based extraction, or b) NPCM (non-polymer cellular material)dissolution-based extraction. NPCM dissolution-based extraction methodsutilize chemicals (such as hypochlorite, or bleach), enzymes (such asprotease), heat (especially to reach temperatures above 100° C.), pH(acids and bases), and/or mechanical means (such as homogenization) tobreak down, oxidize, and/or emulsify non-PHA cellular material. In somecases, extraction methods from both categories can be combined, such asthe simultaneous utilization of hypochlorite and methylene chloride.

NPCM dissolution-based extraction methods require continuous andnon-recoverable chemical inputs, such as hypochlorite, peroxides,enzymes, and pH adjustors, and also generate significant waste disposalissues. Thus, while these methods are effective, the use ofsolvent-based extraction methods is generally preferred in the industrydue to the capacity of solvents to be distilled and recovered forcontinuous re-utilization in a closed loop cycle. Unfortunately, despiteits benefits, some solvent-based extraction methods are energy intensiveprocesses that play a major role in the high cost of PHA production,often accounting for more than 50% of total production costs.Accordingly, there exists a significant need for a novel method tosignificant increase the energy efficiency of solvent-based extraction.

In several embodiments, a process for the extraction ofpolyhydroxyalkanoates from biomass using a solvent-based extractionmethod is provided., wherein the energy required to carry out theprocess is reduced relative to prior solvent-based extraction methods.Specifically, in one embodiment a high efficiency PHA extraction processis provided comprising providing a PHA-containing biomass comprising PHAand water, mixing the biomass with a solvent at a temperature sufficientto dissolve at least a portion of the PHA into the solvent and at apressure sufficient to enable substantially all or part of the solventto remain in liquid phase, thereby producing a PHA-lean biomass phaseand a PHA-rich solvent phase comprising solvent, water, and PHA,separating the PHA-rich solvent phase from the PHA-lean biomass phase ata temperature and pressure sufficient to enable substantially all orpart of the solvent to remain in the liquid phase and preventsubstantially all or part of the PHA within the PHA-rich solvent phasefrom precipitating, reducing the pressure or increasing the temperatureof the PHA-rich solvent phase to cause the solvent to vaporize and thePHA to precipitate or become a solid while maintaining the temperatureand/or the pressure of the PHA-rich solvent phase to prevent all or partof the temperature-dependent precipitation of the PHA into water, andcollecting the solid PHA material, including optionally separating theprecipitated PHA from the solvent and/or the water.

In the past, PHA precipitation has been induced in PHA-rich solvent bya) adding a non-PHA solvent to the solvent phase to reduce thesolubility of PHA in the solvent phase and/or b) reducing thetemperature of the solvent phase to reduce the solubility of PHA in thesolvent. In particular, some methods 1) dissolve PHA in a solvent byincreasing the temperature of the solvent and 2) precipitate PHA byreducing the temperature (and, thus, solubility) of the solvent. Othermethods require adding water to a solution of PHA-rich solventcomprising dissolved PHA, wherein the addition of water to the solutionreduces the solubility of the PHA in the solvent and causes the PHA toprecipitate into the solvent and/or water. (For example, U.S. Pat. No.4,562,245; U.S. Pat. No. 4,968,611; U.S. Pat. No. 5,894,062; U.S. Pat.No. 4,101,533, all herein incorporated by reference.) In each of thesecases, energy efficiency is compromised; specifically, by adding wateror a non-PHA solvent to reduce the PHA solubility of a solvent,additional energy is required for downstream water/non-solvent removal,heating, and/or distillation. By reducing the temperature of the solventto reduce the solubility of the solvent and induce PHA precipitation,heat energy is redundantly expended, as the solvent must be re-heatedfor distillation and recovery. Therefore, in several embodiments, ratherthan adding a non-solvent to a PHA-solvent or reducing the temperatureof the PHA-solvent to effect PHA precipitation, pressure and/or anincrease in temperature is used to induce the precipitation orsolidification of the PHA without redundantly reducing the temperatureof solvent. Thus, in such embodiments, there is a significant reductionin the energy required to heat and/or distill non-solvent and/or solventin downstream PHA processing.

In one embodiment, the extraction process is substantially carried outat intracellular temperatures less than about 100° C. In otherembodiments, temperatures for extraction range from about 10° C. toabout 30° C., from about 30° C. to about 50° C., from about 50° C. toabout 70° C., from about 70° C. to about 90° C., from about 90° C. toabout 120° C., from about 100° C. to about 140° C., from about 20° C. toabout 150° C., or from about 120° C. to about 180° C., or higher. In oneembodiment, cells are reused for polymerization following the extractionprocess as viable cells. In one embodiment, PHA-containing biomass istreated to one or more chemical treatment steps to control, modify, orincrease the concentration or functional characteristics (e.g.,molecular weight, monomer composition, melt flow profile, purity,non-PHA residuals concentration, protein concentration, DNAconcentration, antibody concentration, antioxidant concentration) of PHAin a PHA-containing material or biomass. In one embodiment, temperatureis used to control, modify, reduce, or optimize the molecular weight,polydispersity, melt flow, and other characteristics of PHA. In oneembodiment, temperature and/or time is used to control the molecularweight of PHA between the range of 5,000,000 and 10,000 daltons. In oneembodiment, a slurry comprising PHA-containing biomass and a culturemedia is subject to one or more water removal steps or water additionsteps to increase the concentration of PHA in a PHA-containing biomass.In one embodiment, the water removal step is a dewatering step orcombination of dewatering steps, such as centrifugation, filtration,spray drying, flash drying, and/or chemical dewatering (e.g., withacetone, ethanol, or methanol), wherein at least a portion of the waterconcentration relative to the concentration of PHA-containing biomass inthe slurry is reduced. In one embodiment, a temperature and/or pressurecontrol step is carried out under atmospheric (0 psi), sub-atmospheric(−100-0 psi), or above-atmospheric pressure (e.g., 0-30,000 psi) and attemperature conditions wherein the PHA-containing biomass, or the liquidin and/or around the PHA-containing biomass, is maintained, for at leasta period of time, at a temperature ranging from about −30 to about 10degrees Celsius, about 10 degrees Celsius to about 100 degrees Celsius,about 10 degrees Celsius to about 150 degrees Celsius, about 20 degreesCelsius to about 250 degrees Celsius, and/or about 100 to about 200degrees Celsius. In one embodiment, the PHA-containing biomass issubject to a dewatering step before or after the temperature and/orpressure control step, wherein the dewatering step is centrifugation,filtration, and/or spray drying, to produce a fully or partiallyde-watered PHA-containing biomass or PHA-containing biomass slurry,wherein the water concentration of the dried slurry is less than about99%, less than about 95%, less than about 80%, less than about 60%, lessthan about 40%, less than about 30%, less than about 20%, less thanabout 10%, less than about 5%, less than about 3%, less than about 2%,or less than about 1% water. In one embodiment, the PHA-containingbiomass is subject to a temperature control step, wherein the liquidchemicals within and/or around the biomass, e.g., water, methylenechloride, carbon dioxide, and/or ammonia, is controlled and maintainedat a temperature of at least −30, at least −10, at least −5, at least−4, at least −3, at least −2, at least −1, at least 0, at least 10, atleast 20, at least 30, at least 40, at least 50, at least 60, at least70, at least 80, at least 90, at least 100, at least 110, at least 120,at least 130, at least 140, at least 150, at least 160, at least 170, atleast 180, at least 190, at least 200, at least 210, at least 220, atleast 230, at least 240, at least 250, at least 260, at least 270, atleast 280, at least 290, at least 300 degrees Celsius (or overlappingranges of those temperatures), wherein the solubility, dispersitivity,or homogeneity of non-PHA material increases in the liquid in and/oraround the biomass. In one embodiment, the PHA-biomass is not driedprior to such temperature control step. In one embodiment, thePHA-containing biomass is dried or de-watered prior to such temperaturecontrol step. In one embodiment, the PHA-containing biomass is filteredor centrifuged following the temperature control step. In oneembodiment, the PHA-containing cell slurry is not dewatered, forexample, by centrifugation or other drying mechanism, prior to thetemperature control step. In one embodiment, a mechanism to impart shearonto or into the PHA-containing biomass is coupled with a temperaturecontrol step; such shear may be imparted in the form of one or moreshear induction mechanisms, e.g., centrifugal pump, agitator, blender,high shear mixer, vortex mixer, etc. In one embodiment, thePHA-containing biomass is dewatered in one step and the treatedPHA-containing biomass is further dewatered in one or more additionalsteps. In one embodiment, the PHA-containing biomass is dewatered, waterand/or other chemicals are added and temperature and/or pressure iscontrolled, and the treated PHA-containing biomass is further dewateredand/or purified. In one embodiment, the water and/or chemicals within,around, and/or added to the PHA-containing biomass is temperature and/orpressure controlled, and the treated PHA-containing biomass is furtherpurified in one or more purification steps. In one embodiment, thetemperature control step process time is approximately 1 second,approximately 5 seconds, approximately 10 seconds, approximately 25seconds, approximately 60 seconds, approximately 2 minutes,approximately 5 minutes, approximately 20 minutes, approximately 45minutes, approximately 1 hour, approximately 2 hours, approximately 5hours, approximately 6 hours, approximately 7 hours, approximately 12hours, approximately 15 hours, approximately 24 hours, approximately 36hours, approximately 48 hours, or overlapping ranges of those times. Inone embodiment, inorganic materials may be used to effect PHAmodification, purification, or extraction, including carbon dioxide anddinitrogen. In one embodiment, the PHA-containing slurry or biomass istreated with carbon dioxide under elevated temperatures and pressures,including supercritical ranges, to induce PHA extraction or functionalmodification of PHA. In one embodiment, organic solvents, includingmethylene chloride, acetone, chloroform, dichloroethane, ethanol, and/ormethanol are used in conjunction with any of the above steps. In oneembodiment, solvents or extraction materials may be used or recycled forbiomass production, biogas production, and/or PHA synthesis.

In one embodiment, chemicals are added to a PHA-containing biomass tocause the crystallization of PHA. In one embodiment, methylene chloride,carbon dioxide, acetone, water, dichloroethane, or methanol may be addedto a PHA-containing biomass in order to induce the crystallization ofPHA in the PHA-containing biomass. In some embodiments, this step may beuseful for the downstream processing of PHA, wherein crystallized PHA isless prone than amorphous to degradation, including molecular weightloss, when contacted with extraction chemicals, including solvents,enzymes, acids, bases, and bleach. In one embodiment, silicon, silica,derivatives thereof, and/or chemicals containing silicon may be added tothe PHA-containing biomass in order to impact the metabolic status ofthe culture, and thereby control the functional characteristics of thePHA produced by the culture, including molecular weight, monomercomposition, co-polymer structure, melt index, and polydispersity.

The removal of non-PHA materials from PHA often accounts for asignificant fraction of PHA production costs. As a specific example,pigments often cause unwanted discoloration of PHA, and must be removedthrough costly processes, such as ozonation, peroxide washing, acetonewashing, ethanol washing, solvent refluxing, hypochlorite digestion,enzymatic degradation, surfactant dissolution, or other methodsdisclosed in the art. In several embodiments, a non-sterile process isused to select for microorganisms exhibiting minimal pigmentation.Applicant has surprisingly discovered that, by manipulating theconcentration of dissolved oxygen in a microorganism system, a culturemay be selected wherein white, tan, off-white, light brown, and/or lightyellow pigments are exhibited rather than purple, red, pink, dark brown,orange, or other heavy pigments. Specifically, in some embodiments, anexcess of dissolved oxygen is introduced into a growth media oversuccessive periods, resulting in selective growth of strains ofmethanotrophic microorganisms which produce white, tan, off-white, lightbrown, and light yellow pigments, rather than those producing pink, red,purple, dark yellow, dark brown, and/or other heavy pigments. As aresult, such embodiments, reduce the need for costly downstreampigmentation removal.

As used herein, the term “PHA-reduced biomass” or “substantiallyPHA-reduced biomass” shall be given its ordinary meaning and shall beused to describe a biomass material wherein the concentration of PHArelative to non-PHA material has been reduced in a PHA-containingbiomass through the utilization of a PHA extraction process. In someembodiments, PHA-reduced biomass is further treated in a variety ofways. In some embodiments, the further treatment includes, but is notlimited to, one or more of dewatering, chemical treatment, sonication,additional PHA extraction, homogenization, heat treatment, pH treatment,hypochlorite treatment, microwave treatment, microbiological treatment,including both aerobic and anaerobic digestion, solvent treatment, waterwashing, solvent washing, and/or drying, including simple or fractionaldistillation, spray drying, freeze drying, rotary drying, and/or ovendrying.

In one embodiment, PHA-reduced biomass is substantially dried, whereinthe resulting dried material comprises less than about 99% liquids,including water, solvents, salts, and/or growth-culture media. In someembodiments, the drying processes disclosed herein yield a driedmaterial containing between about 95% and about 75% liquids, betweenabout 75% and about 50% liquids, between about 50% and about 25%liquids, between about 25% and about 15% liquids. between about 15% andabout 10% liquids, between about 10% and about 1% liquids, andoverlapping ranges thereof. In some embodiments, drying is complete(e.g., between about 1% and 0.1% liquids, or less). In anotherembodiment of the invention, a liquid phase comprising PHA-reducedbiomass is subjected to filtration, centrifugation, densitydifferentiation, or other method to increase the solids content of thePHA-reduced biomass.

Traditionally, the separation of biomass from liquid growth media isdifficult and impractical due to the plugging and foulingcharacteristics of biomass. In several embodiments, a method enablingthe efficient filtration of microorganisms is provided. In someembodiments, a liquid chemical is added to the growth media comprisingmicroorganisms, wherein the liquid chemical is ethanol, acetone,methanol, methylene chloride, ketones, alcohols, and/or chlorinatedsolvents, or a combination thereof. In some embodiments, microorganismsare then efficiently separated from liquid growth media using standardfiltration equipment, such as a Buchner filter, filter press, or similarapparatus. In one embodiment, approximately 2 parts acetone are mixedwith one part water, including both intracellular and extracellularwater, to effect the efficient filtration of microorganisms comprisingthe water.

As used herein, the terms “isolated PHA” and “substantially isolatedPHA” shall be given their ordinary meaning and shall refer to PHA thathas been removed from a biomass material as a result of an extractionprocess, or a biomass material wherein the concentration of PHA relativeto non-PHA material has been increased by an extraction process. Inseveral embodiments, isolated PHA is further treated in one or more of avariety of ways, including, but not limited to, purification,filtration, washing, oxidation, odor removal, pigment removal, lipidremoval, non-PHA material removal, and/or drying, includingcentrifugation, filtration, spray drying, freeze drying, simple orfractional distillation, or density differentiation. Methods for thepurification of PHA include the use of peroxides, water, hypochlorite,solvents, ketones, alcohols, and various other agents to separate andremove non-PHA material from PHA material. In some embodiments, PHA isremoved from a microorganism culture by solvent extraction to produceisolated PHA in a PHA-rich solvent phase and PHA-reduced biomass in aPHA-lean liquid phase. In some embodiments, the solvent phase isseparated from the liquid phase by filtration or centrifugation. In someembodiments, both centrifugation and filtration are used in combination(e.g., sequentially). In some embodiments, centrifugation is optionallyfollowed by filtration. In other embodiments, filtration is optionallyfollowed by centrifugation. Filtration, in some embodiments is performedunder vacuum pressure, via gravity feed, under positive pressure, or inspecialized filtration centrifuges. In some embodiments, the filter poresize is adjusted based on the species composition of the microorganismculture. In some embodiments, pore sizes of up to 200 μm are used. Insome embodiments, smaller pore sizes are used, for example 15 to 20 μm,10 to 15 μm, 5 to 10 μm, 1 to 5 μm, 0.001 to 1 μm, and overlappingranges thereof.

In addition to the steps outlined above, additional steps may be takento remove solvent from the extracted PHA, including evaporation, solventcasting, steam stripping, heat treatment, and vacuum treatment, each ofwhich may be preferential, cost-effective, time-effective, oradvantageous depending on the volatility and type of solvent used. Inother embodiments, active processes can be used to reduce the solventcontent of the solvent-PHA mixture. For example, in certain embodiments,alterations in temperature of certain solvents change the solubility ofthe PHA in the solvent, which effectively removes solvent from the PHA(e.g., the solvent is now separable from a precipitated PHA). In someembodiments, filtration, solvent temperatures, or vacuum treatment canbe increased to reduce a portion of the solvent. In some embodiments,solvent to PHA ratios post extraction, filtration, evaporation, solventcasting, steam stripping, heat treatment, and/or vacuum treatment rangefrom about 0.1:1 to about 1,000:1, including about 0.2:1, about 0.3:1,about 4.0:1, about 5.0:1, about 10.0:1, about 20.0:1, about 60:1, about70:1, about 80:1, about 90:1, about 100:1, about 200:1, about 500:1, andabout 900:1.

As a result of the processes disclosed above, in some embodiments, thesolvent is substantially removed from the isolated PHA in the PHA-richsolvent phase and the liquid is substantially removed from thePHA-reduced biomass in the PHA-lean liquid phase. In some embodiments,the isolated PHA is dried in a heated vessel to produce substantiallypure isolated PHA (e.g., at least 80% PHA by dry weight, preferably atleast 98% PHA by weight, more preferably at least 99% PHA by weight).

Numerous varieties of heated or drying vessels may be used to dry theisolated PHA, including ovens, centrifugal dryers, air dryers, spraydryers, and freeze dryers, among others. In some embodiments, heat isapplied to a drying vessel to speed the process and/or to remove (e.g.,evaporate traces of solvent from the PHA). It shall be appreciated thatthe moisture content of the isolated PHA will depend, in someembodiments, on the solvent used, and the corresponding separationtechnique used (as described above). For example, a volatile solvent incombination with ultracentrifugation would result in a less moistextracted PHA, while a less active separation technique (e.g., gravityphase separation) would yield a more moist extracted PHA. In someembodiments, internal dryer temperatures range from 20° C. to 40° C. toabout 200° C. In some embodiments, internal temperatures range fromabout 50° C. to about 90° C., about 90° C. to about 180° C., about 65°C. to about 175° C. and overlapping ranges thereof. In some embodiments,outlet temperatures are substantially lower than inlet on internaltemperatures. In some embodiments, outlet temperatures range from 30° C.to 90° C. In some embodiments, outlet temperatures are between about 35°C. to 40° C., about 40° C. to about 45° C., about 45° C. to about 50°C., about 50° C. to about 55° C., about 55° C. to about 90° C., andoverlapping ranges thereof. It shall also be appreciated that theinternal and outlet temperatures may be adjusted throughout the dryingprocess (e.g., the temperature difference may initially be large, butdecrease over time, or vice versa).

From the above discussion, it shall be appreciated that the type ofdryer used, and the temperatures used (if other than atmospherictemperatures) are easily tailored to correspond to the techniques usedin the extraction process. In some embodiments, particular dryercomponents are beneficial in the isolation of PHA. For example,depending on the moisture content of the extracted PHA (e.g., the amountof residual solvent) particular components of an evaporative-type dryer,such as an oven dryer, rotary dryer, spin flash dryer, spray dryer(equipped with various types of nozzle types, including rotary atomizor,single flow atomizer, mist atomizer, pressure atomizer, dual-flowatomizer) convection heat dryer, tray dryer, scrape-flash dryer, orother dryer type are used. By way of additional example, if a freezedryer (e.g., a lyophilizer) is used, in some embodiments a manifolddryer is used, optionally in conjunction with a heat source. Also by wayof example, a tray lyophilizer can be used, in some embodiments, withthe isolated and dried PHA being stored and sealed in containers (e.g.,vials) before re-exposure to the atmosphere. In certain embodiments,such an approach is used when long-term storage of the PHA is desired.

It shall also be appreciated that certain varieties of heated/dryingapparatuses have adjustable flow rates that can be tailored to themoisture content of the isolated PHA. For example, an isolated PHAhaving a high moisture content would be fed into a dryer at a slowerinput rate to allow a higher degree of drying per unit of PHA inputtedinto the dryer. Conversely, a low moisture content isolated PHA wouldlikely require less time to dry, and therefore could be input at afaster rate. In some embodiments, input rates of isolated PHA range fromseveral hundred liters of isolated PHA-solvent mixture per minute downto several milliliters per minute. For example, input rates can rangefrom about 10 mL/min to about 6 L/min, including about 10 ml/min toabout 50 ml/min, about 50 mL/min to about 100 ml/min, about 100 ml/minto about 500 ml/min, about 500 ml/min to about 1 L/min, about 1 L/min toabout 2 L/min, about 2 L/min to about 4 L/min, about 4 L/min to about 6L/min, and about 100 L/min to about 500 L/min

A range of PHA functional characteristics can be attained by mixing onePHA molecule, such as PHB, with various PHA polymers, including PHB, atvarious molecular weights. Therefore, in several embodiments, a firstisolated PHA is heated to reduce the molecular weight of the firstisolated PHA, and then subsequently mixed with a second PHA wherein themolecular weight of the second PHA is higher than the molecular weightof the first PHA. With such embodiments, Applicant has surprisinglydiscovered methods to functionalize one or more PHAs, including PHB. Inadditional embodiments of the invention, the molecular weight of a firstPHA is reduced from about 800,000 to about 5,000,000 Daltons to about30,000 to about 800,000 Daltons and mixed with a second PHA with amolecular weight of about 800,000 to about 5,000,000 Daltons to modifythe functionalities of the input PHAs. In yet another embodiment, afirst PHA is mixed with a second PHA wherein the molecular weight of thefirst PHA is at least 0.1% less than the molecular weight of the secondPHA. In some embodiments, the difference in molecular weight between thefirst and second PHA is about 0.1% to about 1%, about 1% to about 10%,about 10% to about 20%, about 20% to about 30%, about 30% to about 40%,about 40% to about 50%, about 50% to about 60%, about 60% to about 70%,and overlapping ranges thereof. In still additional embodiments, PHAshaving greater differences in molecular weight are used. In yet anotherembodiment, the molecular weight of a first PHB is reduced to less thanabout 100,000-500,000 Daltons and mixed with a second PHA with amolecular weight greater than about 100,000-500,000 Daltons to modifythe functionality of the input PHB. It shall be appreciated that inputPHA and PHB weight may vary from the ranges disclosed above, but basedon the differences in the molecular weights, the alteration infunctionality of the input PHB is still achieved.

In one embodiment, the invention comprises a PHA comprising carbonderived from PHA-reduced biomass, wherein the PHA-reduced biomasscomprises carbon derived from one or more carbon-containing gas and/orone or more source of source of carbon. In one embodiment, PHA isco-mingled and/or melted with biomass, including, as examples,methanotrophic, autotrophic, heterotrophic biomass, and/or PHA-reducedbiomass, to improve the functional characteristics of the PHA. In oneembodiment, PHA is co-mixed and/or melted with biomass, including, asexamples, methanotrophic, autotrophic, heterotrophic biomass, and/orPHA-reduced biomass, to improve the functional characteristics of PHA.In one embodiment, the percentage of non-PHA microorganism biomassincluded in a PHA, PHA compound, or PHA mixture is about 0.00001% toabout 0.001%, about 0.001% to about 0.01%, about 0.01% to about 0.1%, toabout 0.1% to about 0.5%, about 0.5% to about 1%, about 1%, to about 2%,about 2% to about 3%, about 3% to about 5%, about 5% to about 7%, about7% to about 10%, about 10% to about 15%, about 15% to about 20%, about20% to about 30%, about 30% to about 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 98%, about 98% to about 99.99%, andoverlapping ranges thereof. In some embodiments, the inclusion ofmicroorganism biomass to a PHA improves the functional characteristicsof a PHA by acting as one or more of the following: nucleating agent,plasticizer, compatibilizer, melt flow modifier, mold release agent,filler, strength modifier, elasticity modifier, or density modifier. Insome embodiments, microscopic size of microorganism biomass, includingnucleic acids and proteins, is particularly and surprisingly effectiveas a functionalization agent for PHA. In some embodiments, microorganismbiomass acts as a surprisingly effective compatibilizer for PHA andnon-PHA polymers, such as polypropylene and polyethylene. In oneembodiment, microorganism biomass is mixed with a PHA to modify thenucleation, plasticization, compatibilization, melt flow, density,strength, elongation, elasticity, mold strength, mold release, and/orbulk density characteristics of a PHA, which may be melted, extruded,film blown, die cast, pressed, injection molded, or otherwise processed.In one embodiment, PHA is partially or not removed from PHA-containingmicroorganism biomass prior to melt processing, e.g., extrusion,injection molding, etc. In one embodiment, non-PHA biomass parts ormaterials are caused to remain with PHA derived from a PHA-containingbiomass in order to modify or control the functional characteristics ofa PHA material. In one embodiment, PHA is present in PHA-containingbiomass at a concentration ranging from 1-99.99999%, 1-99.99%, 1-99%,5-99%, 10-99%, 20-99%, 30-99%, 50-99%, 70-99%, 80-99%, 90-99%, 95-99%,or 98-99%, and overlapping ranges thereof. In one embodiment, PHA,biomass, and one or more non-PHA material, polymer, or thermoplastic aremixed, melted, or processed together. In one embodiment, the non-PHApolymer consists of one or more of the following: polypropylene,polyethylene, polystyrene, polycarbonate, Acrylonitrile butadienestyrene, Polyethylene terephthalate, Polyvinyl chloride, Fluoropolymers,Liquid Crystal Polymers, Acrylic, Polyamide/imide, Polyarylate, Acetal,Polyetherimide, Polyetherketone, Nylon, Polyphenylene Sulfide,Polysulfone, Cellulosics, Polyester, Polyurethane, Polyphenylene oxide,Polyphenylene Ether, Styrene Acrylonitrile, Styrene Maleic Anhydride,Thermoplastic Elastomer, Ultra High Molecular Weight Polyethylene,Epoxy, Melamine Molding Compound, Phenolic, Unsaturated Polyester,Polyurethane Isocyanates, Urea Molding Compound, Vinyl Ester,Polyetheretherketone, Polyoxymethylene plastic, Polyphenylene sulfide,Polyetherketone, Polysulphone, Polybutylene terephthalate, Polyacrylicacid, Cross-linked polyethylene, Polyimide, Ethylene vinyl acetate,Styrene maleic anhydride, Styrene-acrylonitrile, Poly(methylmethacrylate), Polytetrafluoroethylene, Polybutylene, Polylactic acid,Polyvinylidene chloride Polyvinyl chloride, Polyvinyl acetate, Polyvinylacetate co-Polyvinylpyrrolidone, Polyvinylpyrrolidone, Polyvinylalcohol, Cellulose, Lignin, Cellulose Acetate Butryate, Polypropylene,Polypropylene Carbonate, Propylene Carbonate, Polyethylene, ethylalcohol, ethylene glycol, ethylene carbonate, glycerol, polyethyleneglycol, pentaerythritol, polyadipate, dioctyl adipate, triacetylglycerol, triacetyl glycerol-co-polyadipate, tributyrin, triacetin,chitosan, polyglycidyl methacrylate, polyglycidyl metahcrylate,oxypropylated glycerine, polyethylene oxide, lauric acid, trilaurin,citrate esters, triethyl citrate, tributyl citrate, acetyl tri-n-hexylcitrate, saccharin, boron nitride, thymine, melamine, ammonium chloride,talc, lanthanum oxide, terbium oxide, cyclodextrin, organophosphoruscompounds, sorbitol, sorbitol acetal, sodium benzoate, clay, calciumcarbonate, sodium chloride, titanium dioxide, metal phosphate, glycerolmonostearate, glycerol tristearate, 1,2-hydroxystearate, celluloseacetate propionate, polyepichlorohydrin, polyvinylphenol, polymethylmethacrylate, polyvinylidene fluoride, polymethyl acrylate,polyepichlorohydrin-co-ethylene oxide, polyvinyl idenechloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, phenolpoly(4,4′-dihydroxydiphenyl ester, and/or other similar materials. Inone embodiment, PHA, methanotrophic, autotrophic, and/or heterotrophicmicroorganism biomass, and a non-PHA polymer are mixed and meltedtogether. In one embodiment, the concentration of non-PHA microorganismbiomass in such a mixture ranges from 0.0001% to 90%, 0.1% to 30%, 0.1%to 10%, or 0.5% to 8%, and overlapping ranges thereof. In oneembodiment, the functional characteristics of a polyhydroxyalkanoate(PHA) material are augmented, controlled, or optimized, comprising thesteps of: (a) providing a PHA and a microorganism biomass, (b) combiningthe PHA and the biomass in a mixture to form a compound, (c) heating thecompound to between 40 degrees Celsius and 250 degrees Celsius. In oneembodiment, the biomass is present in said mixture at a concentration ofabout 0.1 to about 20%. In one embodiment, the biomass is present insaid mixture at a concentration of about 0.1 to about 80%. In oneembodiment, the biomass is present in said mixture at a concentration ofabout 0.1 to about 8%. In one embodiment, the biomass is methanotrophicbiomass. In one embodiment, the biomass is autotrophic biomass. In oneembodiment, the biomass is heterotrophic biomass. In one embodiment, thebiomass is present in said mixture at a concentration of about 0.1 toabout 20%. In one embodiment, the functional characteristics of apolyhydroxyalkanoate (PHA) material are optimized, controlled, oraugmented, comprising the steps of: (a) providing a PHA, a microorganismbiomass, and a non-PHA polymer, (b) combining the PHA, the non-PHApolymer, and the biomass in a mixture to form a compound, and (c)heating the compound to between 40 degrees Celsius and 250 degreesCelsius. In one embodiment, the biomass is present in said mixture at aconcentration of between about 0.1 to about 20%. In one embodiment, thebiomass is present in said mixture at a concentration of between about0.1 to about 80%. In one embodiment, the biomass is present in saidmixture at a concentration of between about 0.1 to about 8%. In oneembodiment, the biomass is methanotrophic biomass. In one embodiment,the biomass is autotrophic biomass. In one embodiment, the biomass isheterotrophic biomass. In one embodiment, the biomass is present in saidmixture at a concentration of between about 0.1 to about 20%. In oneembodiment, the biomass is present in said mixture at a concentration ofbetween about 0.1 to about 20%. In one embodiment, the biomass ispresent in said mixture at a concentration of between about 0.1 to about80%. In one embodiment, the biomass is present in said mixture at aconcentration of between about 0.1 to about 8%. In one embodiment, thebiomass is methanotrophic biomass. In one embodiment, the biomass isautotrophic biomass. In one embodiment, the biomass is heterotrophicbiomass. In one embodiment, the non-PHA polymer is one or more of thefollowing: polypropylene, polyethylene, polystyrene, polycarbonate,Acrylonitrile butadiene styrene, Polyethylene terephthalate, Polyvinylchloride, Fluoropolymers, Liquid Crystal Polymers, Acrylic,Polyamide/imide, Polyarylate, Acetal, Polyetherimide, Polyetherketone,Nylon, Polyphenylene Sulfide, Polysulfone, Cellulosics, Polyester,Polyurethane, Polyphenylene oxide, Polyphenylene Ether, StyreneAcrylonitrile, Styrene Maleic Anhydride, Thermoplastic Elastomer, UltraHigh Molecular Weight Polyethylene, Epoxy, Melamine Molding Compound,Phenolic, Unsaturated Polyester, Polyurethane Isocyanates, Urea MoldingCompound, Vinyl Ester, Polyetheretherketone, Polyoxymethylene plastic,Polyphenylene sulfide, Polyetherketone, Polysulphone, Polybutyleneterephthalate, Polyacrylic acid, Cross-linked polyethylene, Polyimide,Ethylene vinyl acetate, Styrene maleic anhydride, Styrene-acrylonitrile,Poly(methyl methacrylate), Polytetrafluoroethylene, Polybutylene,Polylactic acid, and/or Polyvinylidene chloride, Polyvinyl chloride,Polyvinyl acetate, Polyvinyl acetate co-Polyvinylpyrrolidone,Polyvinylpyrrolidone, Polyvinyl alcohol, Cellulose, Lignin, CelluloseAcetate Butryate, Polypropylene, Polypropylene Carbonate, PropyleneCarbonate, Polyethylene, ethyl alcohol, ethylene glycol, ethylenecarbonate, glycerol, polyethylene glycol, pentaerythritol, polyadipate,dioctyl adipate, triacetyl glycerol, triacetyl glycerol-co-polyadipate,tributyrin, triacetin, chitosan, polyglycidyl methacrylate, polyglycidylmetahcrylate, oxypropylated glycerine, polyethylene oxide, lauric acid,trilaurin, citrate esters, triethyl citrate, tributyl citrate, acetyltri-n-hexyl citrate, saccharin, boron nitride, thymine, melamine,ammonium chloride, talc, lanthanum oxide, terbium oxide, cyclodextrin,organophosphorus compounds, sorbitol, sorbitol acetal, sodium benzoate,clay, calcium carbonate, sodium chloride, titanium dioxide, metalphosphate, glycerol monostearate, glycerol tristearate,1,2-hydroxystearate, cellulose acetate propionate, polyepichlorohydrin,polyvinylphenol, polymethyl methacrylate, polyvinylidene fluoride,polymethyl acrylate, polyepichlorohydrin-co-ethylene oxide, polyvinylidene chloride-co-acrylonitrile, polycyclohexyl methacrylate, celluloseacetate butryate, cellulose, starch, cellulose acetatebutyrate-g-polyethyelene glycol, polyvinylidene chlorideco-acrylonitrile, polyvinyl acetate, polyethylene glycolb-poly(e-caprolactone), R-PHB-OH, S-PHB-OH, polyphenolpoly(4,4′-dihydroxydiphenyl ester, 4-tert-butylphenol, polyglutamate,acrylonitrile-butadiene-styrene, polystyrene, styrene acrylonitrile,polyethylene 2,6-napthalate, polypropylene oxide, polyethyleneterepthalate, polybutylacrylate, poly-y-benzyl-1-glutamate,starch-b-PPG-urethane, ethylene propylene rubber-g-sodium acrylateEPR-g-SA, polypropylene carbonate, polypropylene carbonate-co-polyvinylacetate, natural starch, starch adipate, starch-b-polyester-urethane,starch-b-PEG-urethane, PHBV, polycaprolactone, PLLA, polyoxymethylene,polyvinyl acetate-co-vinyl alcohol, ethylene-propylene rubber,ethylene-vinyl-acetate copolymer, synthetic poly3-hydroxybutyrate,poly-3-hydroxybutyrate-co-poly-3-hydroxyvalerate,poly-3-hydroxypropionate, polybutylene succinate-co-butylene adipate,polybutylene succinate-co-caprolactone, and/or phenolpoly(4,4′-dihydroxydiphenyl ester.

Purifying the Isolated PHA

In some embodiments, isolated PHA is purified to produce a PHA materialthat is substantially pure PHA. In some embodiments, the isolated PHA ispurified to at least 20% pure PHA by dry weight. In some embodiments,the isolated PHA is purified to at least 55% pure PHA by dry weight,while in some embodiments, the isolated PHA is purified to at least 90%pure PHA by dry weight. In additional embodiments, purity of theisolated PHA is between about 90 and 99.9%, including about 91, about92, about 93, about 94, about 95, about 96, about 97, about 98, andabout 99% pure.

In several embodiments, isolated PHA may be recovered by any one, or acombination, of the methods described above, including, but not limitedto: washing, filtration, centrifugation, dewatering, purification,oxidation, non-PHA material removal, solvent removal, and/or drying. Insome embodiments, isolated PHA is recovered according to the manner inwhich it has been removed from the culture. For example, in embodimentsin which solvent-based extraction is employed, a recovery method may beemployed to remove the isolated PHA from the solvent and/or othernon-PHA material. In one embodiment, solvent may be used to extract thePHA, wherein the solvent is then substantially removed from the isolatedPHA by carrying out PHA precipitation and filtration, excess solventdistillation and/or removal, and/or drying, resulting in the recovery ofdry, isolated PHA. In embodiments employing enzyme, surfactant,protonic, hydroxide, and hypochlorite-based extraction techniqueswherein the dissolution of non-PHA material is induced, isolated PHA maybe filtered, washed, separated, centrifuged, and/or dried, resulting inthe recovery of dry, isolated, purified PHA. The resultant PHA, in someembodiments, is further used in downstream processing, includingthermoforming.

Returning PHA-Reduced Biomass to the PHA-Producing Culture to ConvertPHA-Reduced Biomass into PHA

In several embodiments of the invention, the PHA-reduced biomass isreturned to the culture to cause the biomass-utilizing microorganismswithin the culture to convert the carbon within the PHA-reduced biomassinto PHA. By using PHA-reduced biomass as a source of carbon for theproduction of microorganisms in a microorganism fermentation system, aseries of biochemical enzymatic pathways are generated in situ by themicroorganism culture to carry out the metabolic utilization ofPHA-reduced biomass for growth, reproduction, and PHA synthesis.

Without being limited by theory, it is believed that gas-utilizingmicroorganisms and biomass-utilizing microorganisms are able to co-existas a single microorganism consortium because they utilize sources ofcarbon that require distinctly different bioenzymatic assimilationpathways. For instance, while methane metabolism requires the methanemonooxygenase enzyme to catalyze the conversion of methane into methanolfor cellular assimilation, and methane monooxygenase is competitivelyinhibited by a wide range of compounds, it is not inherently deactivatedby high concentrations of cellular biomass, including PHA-reducedbiomass. Similarly, the chlorophyll-based metabolic assimilation systemsrequired for the conversion of carbon dioxide into biomass and PHA arenot inherently deactivated or competitively inhibited by highconcentrations of cellular biomass, including PHA-reduced biomass.Likewise, the enzymatic architecture enabling the metabolic utilization,breakdown, and/or assimilation of PHA-reduced biomass is not inherentlydeactivated or competitively inhibited by high concentrations of methaneand/or carbon dioxide, particularly as the process requires neithermethane monooxygenase nor chlorophyll. Without being limited by theory,Applicant believes that the relatively non-competitive, and in somecases commensal or mutualistic relationships between microorganismsconsuming a carbon-containing gas and a PHA-reduced biomass, make itpossible to create a microorganism culture comprising biomass-utilizingmicroorganisms and gas-utilizing microorganisms, wherein bothcarbon-containing gases and PHA-reduced biomass may be metabolized assimultaneously assimilable sources of carbon.

In the case of autotrophic, methanotrophic, and/or biomass-utilizingmicroorganisms, Applicant has found that a mutualistic,positive-feedback loop relationship can be created in a single (oroptionally multiple) reaction vessel. In such embodiments, the oxygencreated by autotrophic metabolism is utilized by methanotrophic and/orbiomass-utilizing microorganisms for metabolic functions, the carbondioxide created by methanotrophic and/or biomass-utilizing microorganismmetabolism is utilized for autotrophic metabolism, the methane and/orcarbon dioxide created by anaerobic methanogenic microorganisms isutilized by methanotrophic microorganisms, and the biomass created bymethanotrophic, autotrophic, and/or heterotrophic microorganisms is usedto provide a source of carbon to methanogenic and/or other heterotrophicmicroorganisms. Due to the microscopic-level induction of oxygen and/orcarbon dioxide created therein, mass transfer efficiencies in severalembodiments are significantly improved over traditional gas inductionmeans, such as gas sparging, mechanical mixing, static mixing, or othermeans known in the art. To applicant's knowledge, prior to thedisclosure herein, the use of autotrophic microorganisms cultured inassociation with heterotrophic microorganisms has never been suggestedas a means to improve mass transfer efficiencies, supply oxygen, and/oraugment microorganism growth rates in a positive feedback loop system.

In several further embodiments of the invention, PHA-reduced biomass isused by heterotrophic microorganisms, including acidogenic, acetogenic,and methanogenic microorganisms, to produce methane, which is furtherutilized by methanotrophic microorganisms to produce biomass, includingPHA. In some embodiments of the invention, anaerobic microorganismscoexist with aerobic microorganisms under microaerobic conditions (e.g.,mean dissolved oxygen concentrations approximately 0.00-1.0 ppm,including about 0.001 to about 0.002 ppm, about 0.002 to about 0.03 ppm,about 0.03 to about 0.04 ppm, about 0.04 to about 0.5 ppm, about 0.5 toabout 0.6 ppm, about 0.6 to about 0.7 ppm, about 0.7 to about 0.8 ppm,about 0.8 to about 0.9 ppm, about 0.9 to about 1.0 ppm, and overlappingranges thereof.

In some embodiments of the invention, heterotrophic, methanotrophic,methanogenic, and/or autotrophic microorganisms are divided intomultiple stages and vessels, in particular, anaerobic and aerobicstages, in order to carry out the conversion of PHA-reduced biomass intomethane and PHA. In further embodiments of the invention, PHA-reducedbiomass is returned to the culture using one or more vessels, whereby itis first converted to carbon dioxide, methane, and/or volatile organiccompounds by a heterotrophic, e.g., methanogenic, microorganismconsortium under anaerobic conditions and then converted to PHA bymethanotrophic microorganisms under aerobic conditions, whereby carbondioxide is also metabolized or otherwise used by autotrophicmicroorganisms, methanotrophic microorganisms, and heterotrophicmicroorganisms.

In several embodiments, light intensity is utilized to regulate thegrowth rate of heterotrophic and/or methanotrophic microorganisms. Insome embodiments, light intensity is manipulated to regulate thegeneration of oxygen by autotrophic microorganisms. In some embodiments,the rate of oxygen generated by autotrophic microorganisms issubsequently used to control the growth and metabolism of heterotrophicand methanotrophic microorganisms.

In several embodiments, carbon dioxide is supplied to autotrophicmicroorganisms in the form of carbon dioxide created by methanotrophicand/or heterotrophic microorganisms. In some embodiments, each of thesevarieties of microorganism is cultured in a single vessel. In someembodiments, methane, sugar, biomass, and/or another non-carbon dioxidesource of carbon is used to grow autotrophic microorganisms. Toapplicant's knowledge, autotrophic microorganisms have never beencultured using methane as a sole carbon input.

Some gas-utilizing microorganisms are unable to produce highconcentrations of intracellular PHA. However, according to severalembodiments, certain microorganism consortiums utilizing PHA-reducedbiomass, or derivatives thereof, as a source of carbon are able togenerate high intracellular PHA concentrations and thus effectivelyconvert low PHA concentration biomass derived from a carbon-containinggas into a high PHA concentration biomass material under the conditionsdisclosed herein. In several embodiments, the concentration (by weight)of intracellular PHA is between about 10% to about 30%, about 30% toabout 50%, about 50% to about 70%, about 70% to about 80%, about 80% toabout 90%, about 90% or more, and overlapping ranges thereof. Thus, inone embodiment, the culture is contacted with the PHA-reduced biomassand then manipulated, according to the processes described herein, toeffect PHA synthesis, wherein the PHA-reduced biomass is converted intoPHA by biomass-utilizing microorganisms. In some embodiments, PHAsynthesis is induced by nutrient limitation, nutrient excess, nutrientimbalance, or large shifts in nutrient concentration. In still furtherembodiments, PHA synthesis is induced by reducing the availability ofnitrogen, oxygen, phosphorus, or magnesium to the culture. In someembodiments, these nutrients are simultaneously reduced (to varying orsimilar degrees). In some embodiments, the nutrients are reducedsequentially. In some embodiments, only one of the nutrients is reduced.For example, in certain embodiments, PHA synthesis is induced byreducing the availability of oxygen to the culture. In some embodiments,this is achieved by manipulating the flow rate of air or oxygen into thegrowth medium. In some embodiments, manipulation of the flow rate ofother carbon-containing gases, such as methane and/or carbon dioxide,into the growth medium, or otherwise manipulating the rate of gastransfer in a system (e.g., by adjusting mixing rates or light injectionrates) is employed. In one embodiment, oxygen limitation is induced byreducing the flow rate of oxygen into the growth medium. In anotherembodiment, oxygen limitation is induced by reducing the rate of lighttransmission into the medium to reduce the production of oxygen byautotrophic microorganisms. In some embodiments of the invention, theconcentration of PHA generated in a biomass-utilizing microorganismculture utilizing PHA-reduced biomass as a source of carbon is at least5%, at least 20%, or at least 50% by dry cell weight; in particularlypreferred embodiments of the invention, the concentration of PHA in abiomass-utilizing microorganism is at least 80% by dry cell weight.

In some embodiments, a PHA-reduced biomass recycling system is utilizedwherein substantially all (e.g., at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 98%) of the PHA-reducedbiomass produced is contacted with the culture until it is convertedinto PHA. In some such embodiments, solid sources of carbon aresubstantially output from the process or culture only in the form ofisolated PHA.

In several embodiments, as carbon-containing gases are continually addedto the medium to effect the production of biomass, the process disclosedabove is repeated. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the culture is removed from the medium,PHA is extracted from the PHA-containing biomass, PHA-reduced biomass isseparated from isolated PHA, isolated PHA is recovered, purified, anddried, and PHA-reduced biomass is sent back to the culture and convertedby the culture into PHA. In one embodiment, substantially allPHA-reduced biomass produced is contacted with the culture until it isconverted into isolated PHA, and solid sources of carbon aresubstantially output from the process only in the form of isolated PHA.In other embodiments, carbon is substantially output from the systemonly in the form of PHA and methane, carbon dioxide, and/or volatileorganic compounds.

The following example is provided to further illustrate certainembodiments within the scope of the invention. The example is not to beconstrued as a limitation of any embodiments, since numerousmodifications and variations are possible without departing from thespirit and scope of the invention.

Example 1

A fermentation system comprising one or more vessels are partiallyfilled with one or more liquid growth mediums, wherein the mediumcomprises methanotrophic, autotrophic, methanotrophic, and/or otherheterotrophic or biomass-utilizing microorganisms containing PHA, and,per liter of water, 0.7-1.5 g KH₂PO₄, 0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃,0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28 mg CaCl₂*2H₂O, 5.0-5.4 mg EDTANa₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O, 0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mgMnCl₂*2H₂O, 0.06-0.08 mg ZnCl₂, 0.05-0.07 mg H₃BO₃, 0.023-0.027 mgNiCl₂*6H₂O, 0.023-0.027 mg NaMoO₄*2H₂O, 0.011-0.019 mg CuCl₂*2H₂O. Oneor more of the mediums are anaerobic and/or aerobic, and carboncontaining gases, including methane, carbon dioxide, and volatileorganic compounds, as well as optionally air or oxygen, are fed into allor part of the system to induce the growth and reproduction ofmicroorganisms through the utilization of carbon-containing gases, aswell as the production of PHA.

Next, a portion of the media volume of the fermentation system is passedthrough a basket centrifuge to increase the solids content of the mediumto approximately 167 g/L. The solids-containing centrate phase of thecentrifuged solution is then transferred to a PHA extraction vessel, andthe substantially solids-free filtrate phase of the centrifuged solutionis recycled back to the fermentation system.

In some embodiments, the solids-containing centrate phase is optionallychemically pre-treated prior to extraction. In some embodiments, one ormore of acids, bases, chloride, ozone, and hydrogen peroxide is added.In several embodiments, chemical pre-treatment increases the efficiencyand yield of the subsequent extraction process. In some embodiments, thechemical pre-treatment functions to break down the cell well (partiallyor fully), thereby liberating a greater portion of the intracellularPHA. In some embodiments, chemical pre-treatment dissolves and/orremoves impurities that negatively impact the PHA extraction process. Insome embodiments, chemical pre-treatment enhances cell agglomeration,which increases the percentage of microorganisms that are extracted(e.g., cells in an agglomerated mass are not separated or lost intransfer steps). Next, following optional chemical pre-treatment,solvent is added into the PHA extraction vessel to create a solventsolution, and the solvent solution is then mixed for a period of time tocause the PHA in both the microorganisms to dissolve into the solvent,and thereby create PHA-rich solvent and PHA-reduced biomass. Over thecourse of a defined mixing period (e.g., 0.1-10 hours), the PHA contentof the microorganisms is reduced by about 80% as it is dissolved intothe solvent.

Next, the solvent solution comprising the PHA-rich solvent andPHA-reduced biomass is passed through a filter located at the bottom ofthe PHA extraction vessel, and the PHA-rich solvent is thereby separatedfrom the PHA-reduced biomass. Water is then added to the PHA extractionvessel to create a water-biomass solution, and the water-biomasssolution is then heated to 75° C. to cause any remaining solventassociated with the PHA-reduced biomass to exit the PHA extractionvessel as a gaseous vapor. The vapor discharged from the PHA extractionvessel is then passed through a heat exchanger and recovered as liquidsolvent. Meanwhile, the PHA-rich solvent is transferred to a PHApurification vessel and mixed with room temperature water to create awater-solvent solution. The water-solvent solution is then heated tocause i) the solvent to exit the PHA extraction vessel as a gaseousvapor and ii) the isolated PHA to precipitate into the water and/orbecome a solid. The solvent vapor created by heating the water-solventsolution is then passed through a heat exchanger and converted intoliquid solvent.

The isolated PHA is then substantially dewatered by filtration in aNutsche filter, and the Nutsche filter containing the substantiallydewatered isolated PHA is then heated to remove any additional volatilecompounds, including trace water and/or solvent. Following heat dryingin the Nutsche filter, the isolated PHA is recovered as substantiallypure PHA (e.g., greater than about 90% PHA).

Concurrently, the water-biomass solution comprising PHA-reduced biomassand water is transferred from the PHA extraction vessel back into thefermentation system, where the PHA-reduced biomass is contacted with oneor more of the microorganism cultures. Next, the medium of thefermentation system is manipulated to cause the one or moremicroorganisms within the system to metabolize the PHA-reduced biomassas a source of carbon and nutrients.

Depending on the embodiment, the culture conditions are adjusted todetermine the point at which the inception of the growth or PHAmetabolism phase occurs. As discussed herein, manipulation of theconcentration of one or more growth culture media nutrients can alterthe metabolic pathways favored by certain microorganisms. Additionally,the use of PHA-reduced biomass-derived carbon for the production ofadditional biomass versus the production of PHA can be tailored based onwhether the intent is to grow the culture (e.g., increase the overallbiomass) or to harvest PHA (e.g., shift the culture from growth phase toproduction of PHA). As such, the reduction, increase, or adjustment ofthe concentration certain growth nutrients, and the timing of suchadjustment, plays a role in the metabolic state and PHA production ofthe culture. Adjustment of growth nutrients can occur at any point afterthe PHA-reduced biomass is returned to the microorganism system. In someembodiments, adjustment is immediate (e.g., within minutes to a fewhours). In some embodiments, a longer period of time elapses. In someembodiments, adjustment in one or more growth nutrients occurs afterabout 2 to 4 hours, after about 4 to 6 hours, after about 6 to 8 hours,after about 8 to 10 hours, after about 10 to 12 hours, after about 12 to14 hours, after about 14 to 18 hours, after about 18 to 24 hours, andoverlapping ranges thereof. In still additional embodiments, adjustmentin one or more growth nutrients occurs after about 2 to about 5 days,about 5 to about 10 days, about 10 to about 15 days, about 15 to about20 days, about 20 to about 30 days, about 30 to about 50 days, andoverlapping ranges thereof. In some embodiments, longer times elapseprior to adjusting one or more growth nutrients to induce PHApolymerization.

After a desired period of time has elapsed, the dissolved oxygen and/ornitrogen concentration (or concentration of another nutrient) of one ormore parts of the medium is reduced or adjusted to cause one or more ofthe microorganisms within the system to utilize the PHA-reduced biomassin the medium as a source of carbon for the synthesis of PHA. In someembodiments, the percent adjustment ranges from about 20% to about 100%,including about 20% to about 30%, about 30% to about 40%, about 40% toabout 50%, about 50% to about 60%, about 60% to about 70%, about 70% toabout 80%, about 80% to about 90%, about 90% to about 100%, andoverlapping ranges thereof. It shall be appreciated that, depending onthe characteristics of a culture in a given embodiment, a specificpercentage reduction, increase, or adjustment in nutrient may not benecessary, but a reduction, increase, or adjustment is used that issufficient to convert certain cells from a relative growth phase to arelative PHA synthesis phase. After approximately 12-24 hours of PHAsynthesis, substantially all of the PHA-reduced biomass within thegrowth medium has been metabolized into biomass-utilizing microorganismbiomass, including PHA. It shall be appreciated that, in certainembodiments, greater or lesser PHA synthesis times result in varyingpercentages of the PHA-reduced biomass within the growth medium beingmetabolized into biomass, including PHA.

As carbon containing gases are continually added to the fermentationsystem to effect the production of biomass, the process is repeated,wherein solid sources of carbon substantially exit the system only inthe form of PHA. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the fermentation vessel is passedthrough a dewatering centrifuge to increase the solids content of thePHA-containing biomass, PHA is extracted from the removed PHA-containingbiomass using a solvent-based extraction system to create PHA-reducedbiomass and isolated PHA, PHA-reduced biomass is separated from isolatedPHA, isolated PHA is recovered, purified, and dried, and PHA-reducedbiomass is sent back to the fermentation system and converted bymicroorganisms into PHA, such that substantially all PHA-reduced biomassproduced is contacted with the culture until it is converted intoisolated PHA, and wherein solid sources of carbon are substantiallyoutput from the process only in the form of isolated PHA.

While the above description of several compositions, systems, andmethods contains many specificities, it should be understood that theembodiments of the present invention described above are illustrativeonly and are not intended to limit the scope of the invention. Numerousand various modifications can be made without departing from the spiritof the embodiments described herein. Accordingly, the scope of theinvention should not be solely determined by the embodiments describedherein, but also by the appended claims and their legal equivalents.

What is claimed is:
 1. A method for enhancing polyhydroxyalkanoate (PHA)generation in a methanotrophic culture, the method comprising: (a)contacting a culture of methanotrophic microorganisms with a mediumcomprising copper, one or more additional nutrients, and acarbon-containing gas that can be metabolized by said culture; (b)incubating said culture in said medium to cause growth of said culture;and (c) inducing a selection pressure in said culture to cause saidculture to generate PHA by: (i) reducing the concentration of copper insaid medium to enable production of soluble methane monooxygenase (sMMO)in species of methanotrophic microorganisms capable of producing sMMOand particulate methane monooxygenase (pMMO) in species ofmethanotrophic microorganisms capable of producing pMMO; (ii) reducingthe concentration of said one or more nutrient in said medium to causesaid culture to generate PHA from said carbon-containing gas, (iii)returning the culture resulting from step c (ii) to the growthconditions of step (b); and (iv) repeating steps (i), (ii) and (iii) atleast two times, to generate a culture of microorganisms that metabolizesaid carbon-containing gas to produce PHA.
 2. The method of claim 1,wherein the concentration of said copper in said medium is reduced to aconcentration less than 0.001 mg/L.
 3. The method of claim 1, whereinthe concentration of said copper in said medium is reduced to be lessthan 0.5 micromolar.
 4. The method of claim 1, wherein the concentrationof said copper in said medium is reduced to be less than 0.1 mg per dryweight gram of said microorganisms.
 5. The method of claim 1, whereinthe concentration of said copper in said medium is reduced to be lessthan 100 mg/L.
 6. The method of claim 1, wherein the concentration ofsaid copper in said medium is reduced to be less than 1 mg/L.
 7. Themethod of claim 1, wherein said reduction of said nutrient comprises adepletion of nitrogen from said media by about 50% or more.
 8. Themethod of claim 1, wherein said carbon-containing gas comprises methane.9. The method of claim 1, wherein said carbon-containing gas comprisescarbon dioxide.
 10. The method of claim 1, wherein said transformedculture produces PHA at a concentration of at least 70% of the drybiomass weight of said microorganisms.
 11. A method for enhancingparticulate methane monooxygenase (pMMO)-based polyhydroxyalkanoate(PHA) production comprising (a) contacting a culture of methanotrophicmicroorganisms with a medium comprising copper and nitrogen; (b)incubating said culture in said medium to cause growth of said cultureand contacting said culture with a carbon-containing gas that can bemetabolized by said culture; and (c) transforming said culture into aculture that generates PHA by: (i) reducing the concentration of copperin said medium to enable production of soluble methane monooxygenase(sMMO) in species of methanotrophic microorganisms capable of producingsMMO and particulate methane monooxygenase (pMMO) in species ofmethanotrophic microorganisms capable of producing pMMO, (ii) reducingthe concentration of nitrogen in said medium to cause said culture togenerate PHA from said carbon-containing gas, (iii) returning theculture to the growth conditions of step (b); and (iv) repeating steps(i), (ii) and (iii) at least two times, wherein said repetitionsselectively favor growth of microorganisms that metabolize saidcarbon-containing gas.
 12. The method of claim 11, wherein saidconcentration of copper in said medium is reduced to a concentrationless than about 0.001 mg/L.
 13. The method of claim 11, wherein saidconcentration of nitrogen in said medium is reduced by about 50%. 14.The method of claim 11, wherein said microorganisms having higherintracellular concentrations of PHA comprise PHA at a concentration ofat least about 70% of the dry biomass weight of the microorganisms. 15.The method of claim 11, further comprising contacting the culture withone or more additional carbon-containing materials selected from thegroup consisting of one or more methanol, acetone, formaldehyde,propane, and ethane.